Nectin-4 antibody conjugates and uses thereof

ABSTRACT

The present disclosure provides conjugates of anti-Nectin-4 antibodies or antigen binding fragments thereof to a myeloid cell agonist, compositions comprising the conjugates, and methods of treating cancer with the conjugates. The present disclosure also provides for anti-Nectin-4 antibodies or antigen binding fragments thereof and method for using the antibodies or antigen binding fragments thereof in treating cancer.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 860234_402_SEQUENCE_LISTING.txt. The text fileis 45.6 KB, was created on Feb. 19, 2021, and is being submittedelectronically via EFS-Web.

BACKGROUND

One of the leading causes of death in the United States is cancer.Conventional methods of cancer treatment, like chemotherapy, surgery, orradiation therapy, tend to be either highly toxic or nonspecific to acancer, or both, resulting in limited efficacy and harmful side effects.However, the immune system has the potential to be a powerful, specifictool in fighting cancers. In many cases tumors can specifically expressgenes whose products are required for inducing or maintaining themalignant state. These proteins may serve as antigen markers fordeveloping more specific anti-cancer treatments that can harness thepower of both innate and adaptive immune responses. The activation ofthese immune responses (e.g., myeloid cell activation) in the tumormicroenvironment and lymphoid structures only has the potential to be apowerful anti-cancer treatment that can be more effective thanconventional methods of cancer treatment with fewer side effects.

BRIEF SUMMARY

In one aspect, the present disclosure provides a myeloid cell agonistconjugate comprising: (a) an anti-Nectin-4 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variableregion (VH) and a light chain variable region (VL), wherein the VHcomprises a CDR1 (VH-CDR1) comprising the amino acid sequence of SEQ IDNOT, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2, aVH-CDR3 comprising the amino acid sequence of SEQ ID NOG; and the VLcomprises a CDR1 (VL-CDR1) comprising the amino acid sequence selectedfrom any one of SEQ ID NOS:4-6, a VL-CDR2 comprising the amino acidsequence of SEQ ID NO:7, and a VL-CDR3 comprising the amino acidsequence of SEQ ID NO:8; (b) a myeloid cell agonist; and (c) a linkercovalently attached to the myeloid cell agonist and the antibody.

In certain embodiments, the conjugate is represented by Formula (I):

wherein: A is the anti-Nectin-4 antibody or antigen-binding fragmentthereof, L is the linker; D_(x) is the myeloid cell agonist, wherein themyeloid cell agonist is a TLR8 agonist; n is selected from 1 to 20; andz is selected from 1 to 20.

In a related aspect, the present disclosure provides a myeloid cellagonist conjugate or salt thereof represented by the formula:

wherein Antibody is an anti-Nectin-4 antibody comprising light chainCDR1, CDR2 and CDR3 set forth in the light chain variable region aminoacid sequence of SEQ ID NO:14 or 13, and heavy chain CDR1, CDR2 and CDR3set forth in the heavy chain variable region amino acid sequence of SEQID NO: 10, as determined by the Kabat index, and L³-D is a linker-TLR8agonist and has the structure:

wherein RX* is a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of the antibody construct, wherein

on RX* represents the point of attachment to a cysteine residue of theantibody construct.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a myeloid cell agonist conjugate disclosed hereinand a pharmaceutically acceptable excipient.

In a further aspect, the present disclosure provides a method oftreating cancer (e.g., a Nectin-4-expressing cancer), comprisingadministering to a subject in need thereof an effective amount of amyeloid cell agonist conjugate or a pharmaceutical composition disclosedherein.

In certain embodiments, the myeloid cell agonist conjugate or thepharmaceutical composition is subcutaneously administered.

In certain embodiments, the effective amount of the myeloid cell agonistconjugate is about 0.1 to about 100 mg/kg, preferably about 0.1 to about25 mg/kg, and more preferably about 0.5 to about 20 mg/kg, per treatmentcycle or per administration.

In certain embodiments, the myeloid cell agonist conjugate or thepharmaceutical composition is subcutaneously administered, and theeffective amount of the myeloid cell agonist conjugate is about 0.1 toabout 100 mg/kg, preferably about 0.1 to about 25 mg/kg, and morepreferably about 0.5 to about 20 mg/kg, per treatment cycle or peradministration.

In another aspect, the present disclosure provides an isolatedmonoclonal antibody, or an antigen-binding fragment thereof, thatspecifically binds to Nectin-4, wherein the antibody or antigen-bindingfragment thereof comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises a heavy chainCDR1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO:1, aVH-CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH-CDR3comprising the amino acid sequence of SEQ ID NO:3; and the VL comprisesa CDR1 (VL-CDR1) comprising the amino acid sequence selected from anyone of SEQ ID NOS:4-6, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence of SEQ IDNO:8.

In a further aspect, the present disclosure provides a conjugatecomprising an anti-Nectin-4 antibody disclosed herein and a smallmolecule drug, such as a TLR8 agonist.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising an anti-Nectin-4 antibody disclosed herein or aconjugate comprising such an antibody and a small molecule drug and apharmaceutically acceptable carrier.

In another aspect, the present disclosure provides an isolated nucleicacid that encodes an anti-Nectin 4 antibody or the heavy chain, thelight chain, the heavy chain variable region, or the light chainvariable region of the anti-Nectin 4 antibody. The present disclosurealso provides a vector comprising the isolated nucleic acid, an isolatedhost cell comprising the isolated nucleic acid or the vector, anisolated host cell that expresses an anti-Nectin 4 antibody disclosedherein, and a method of producing an anti-Nectin-4 antibody comprisingculturing the host cell disclosed herein under conditions suitable forexpressing the antibody.

In a further aspect, the present disclosure provides a method oftreating a Nectin-4-expressing cancer comprising administering to asubject having a Nectin-4-expressing cancer an effective amount of ananti-Nectin-4 antibody, a conjugate comprising an anti-Nectin-4 antibodyand a small molecule drug, or a pharmaceutical composition of comprisingthe antibody or the conjugate disclosed herein.

In certain embodiments, the conjugate comprising an anti-Nectin-4antibody and a small molecule drug, or the pharmaceutical compositionthereof, is subcutaneously administered.

In certain embodiments, the effective amount of the conjugate is about0.1 to about 100 mg/kg, preferably about 0.1 to about 25 mg/kg, and morepreferably about 0.5 to about 20 mg/kg, per treatment cycle or peradministration.

In certain embodiments, the conjugate or the pharmaceutical compositionis subcutaneously administered, and the effective amount of theconjugate is about 0.1 to about 100 mg/kg, preferably about 0.1 to about25 mg/kg, and more preferably about 0.5 to about 20 mg/kg, per treatmentcycle or per administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show that anti-Nectin-4 antibodies and anti-Nectin-4antibody-TLR8 agonist immunoconjugates bind to (A) HEK-293 cellstransfected with human Nectin-4 (FIG. 1A); (B) HEK-293 cells transfectedwith cynomolgus Nectin-4 (FIG. 1B); and (C) MDA-MB-175-VII cells (FIG.1C), which are a Nectin-4 expressing tumor cell line.

FIGS. 2A-2B show TNF-α production in PBMC-Nectin-4+ tumor cellco-cultures when contacted with anti-Nectin-4-TLR8 agonist conjugates,but not when contacted with unconjugated Nectin-4-specific antibodies.PBMCs were cultured 24 hours with (A) Nectin-4-expressing MDA-MB-175-VIIcells (FIG. 2A) or (B) Nectin-4-negative HEK-293 cells (FIG. 2B) in thepresence of equivalent titrated concentrations of anti-Nectin-4-TLR8agonist, matched unconjugated anti-Nectin-4 mAb, or isotype controlantibody conjugate.

FIGS. 3A-3B show that anti-Nectin-4-TLR7 agonist immunoconjugate (asurrogate for human TLR8 agonist conjugates of this disclosure sinceonly TLR7 (and not TLR8) is expressed in murine myeloid cells) caninduce TNF-α production in murine bone marrow-derived macrophages (BMDM)when co-cultured with HEK-293 human expressing Nectin-4.

FIG. 4 is a Kaplan-Meier plot of mice bearing human Nectin-4 expressingEMT6 tumors treated with a surrogate of the Nectin-4 conjugates of thisdisclosure.

FIGS. 5A and 5B show that in vivo treatment with a surrogate for humanNectin4-TLR8 conjugate in tumor-bearing mice leads to increasedintra-tumoral chemokines (FIG. 5A) and cytokines (FIG. 5B). Mice bearingNectin4-expressing EMT6 tumors were treated with a single dose of eitherD6C mIgG2a (unconjugated control) or D6C mIgG2a-Compound 4.1 conjugateand tumors were: (A) harvested at Day 2 and levels of the indicatedchemokines in the tumors were assessed (FIG. 5A), or (B) harvested atDay 5 and levels of the indicated cytokines in the tumors were assessed(FIG. 5B). Statistical significance was determined by Mann-Whitney test.***p<0.001, **p<0.01, *p<0.05.

FIG. 6 shows that the binding of TIGIT-Fc to Nectin4-expressing tumorcells was blocked by the binding domain of hzD6.2C.

FIGS. 7A to 7C depict tumor volume (in cubic millimeters) as a functionof time (measured in days post-treatment) following a subcutaneousinjection into mice bearing human Nectin-4 expressing EMT6 tumors of amouse IgG2a isotype control (mIgG2a) (FIG. 7A), an anti-Nectin-4antibody alone (D6C mIgG2a) (FIG. 7B), or an anti-Nectin-4-TLR7 agonistconjugate surrogate (D6C mIgG2a-Compound 4.1) (FIG. 7C).

FIG. 8 depicts the mean pre-dose and peak serum MCP-1 and IP-10concentrations as compared to dose level in monkeys followingsubcutaneous administration of an anti-Nectin-4-TLR8 agonist conjugate(D6.2C IgG1-Compound 2.14).

DETAILED DESCRIPTION

The present disclosure provides anti-Nectin-4 antibodies, myeloid cellagonist conjugates comprising anti-Nectin-4 antibodies, andpharmaceutical compositions that comprise such antibodies andconjugates. The antibodies, conjugates and pharmaceutical compositionsdisclosed herein are useful in treating cancer alone or in combinationwith other anti-cancer therapeutic agents.

Anti-Nectin-4 antibodies and myeloid cell agonist conjugates comprisingsuch antibodies as provided herein are capable of specifically bindingto Nectin-4 expressing cells. The myeloid cell agonist conjugates arealso capable of inducing TNF-α production from human peripheral bloodmononuclear cells (PBMCs) in the presence of Nectin-4 expressing tumorcells, which indicates that the myeloid cells are being activated by theconjugates of this disclosure. Surprisingly, certain exemplaryconjugates comprising humanized anti-Nectin-4 antibodies not only weremore potent in inducing TNF-α production from PBMCs, but also reached ahigher maximal TNF-α production level compared to conjugates comprisingthe parent anti-Nectin-4 antibody and compared to conjugates comprisinga reference anti-Nectin-4 antibody that cross-blocks the anti-Nectin-4antibodies of this disclosure.

Anti-Nectin-4 antibodies and myeloid cell agonist conjugates comprisingsuch antibodies as provided herein are also capable of increasingintra-tumoral levels of chemokines and cytokines, indicating that theyare capable of enhancing innate immune response driven by myeloid cellactivation, which in turn are capable of nucleating an adaptive immuneresponse by indirectly activating T and NK cells with the tumor.

In certain embodiments, anti-Nectin-4 antibodies and myeloid cellagonist conjugates comprising such antibodies as provided herein arecapable of blocking the binding of TIGIT to Nectin-4 expressed on tumorcells.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

As used in the specification and claims, the singular form “a,” “an,”and “the” includes plural references unless the context clearly dictatesotherwise. It should be understood that the terms “a” and “an” as usedherein refer to “one or more” of the enumerated components.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the terms “include” and “comprise” are used synonymously.

The phrase “at least one of” when followed by a list of items orelements refers to an open ended set of one or more of the elements inthe list, which may, but does not necessarily, include more than one ofthe elements.

The term “about” as used herein in the context of a number refers to arange centered on that number and spanning 15% less than that number and15% more than that number. The term “about” used in the context of arange refers to an extended range spanning 15% less than that the lowestnumber listed in the range and 15% more than the greatest number listedin the range.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include any value(including integers or fractions) or subrange within the recited rangeunless otherwise indicated.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive toward, aspecific antigen. An antibody can include, for example, polyclonal,monoclonal, and genetically engineered antibodies, and antigen bindingfragments thereof. An antibody can be, for example, murine, chimeric,humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody.

As used herein, an “antigen-binding domain” or “antigen-binding fragmentrefers to a region of a molecule that specifically binds to an antigen.An antigen binding domain can be an antigen-binding portion of anantibody or an antibody fragment. An antigen-binding fragment caninclude, for example, a Fab′, F(ab′)₂, Fab, Fv, rIgG, scFv, hcAbs (heavychain antibodies), a single domain antibody, V_(HH), V_(NAR), sdAbs, ornanobody.

As used herein, an “Fc domain” refers to a domain from an Fc portion ofan antibody that can specifically bind to an Fc receptor, such as an Fcγreceptor or an FcRn receptor.

As used herein, “identical” or “identity” refer to the similaritybetween a DNA, RNA, nucleotide, amino acid, or protein sequence toanother DNA, RNA, nucleotide, amino acid, or protein sequence. Identitycan be expressed in terms of a percentage of sequence identity of afirst sequence to a second sequence. Percent (%) sequence identity withrespect to a reference DNA sequence can be the percentage of DNAnucleotides in a candidate sequence that are identical with the DNAnucleotides in the reference DNA sequence after aligning the sequences.Percent (%) sequence identity with respect to a reference amino acidsequence can be the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in thereference amino acid sequence after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. As used herein, the percent sequence identityvalues is generated using the NCBI BLAST 2.0 software as defined byAltschul et al., “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs,” Nucleic Acids Res. 2007, 25,3389-3402, with the parameters set to default values.

A “small molecule” is an organic compound with a molecular weight ofless than 1500, or 100, or 900, or 750, or 600, or 500 Daltons. A “smallmolecule drug” is a small molecule that has a therapeutic effect such astreating a disease or disorder. In some embodiments, a small moleculedrug is a small molecule agonist that has an octanol-water partitioncoefficient (log P) in the range of from 3 to 6, or from 4 to 5, or from2 to 4. In some embodiments, a small molecule agonist has a polarsurface area of less than 200, or less than 150 Å². In some embodiments,the small molecule agonist has not more than five, or not more thanthree, hydrogen bond donors, and not more than 10, or not more thanthree hydrogen bond acceptors. A small molecule is not a protein, apolysaccharide, or a nucleic acid. A “small molecule inhibitor” is asmall molecule that inhibits the activity of another molecule, such as aprotein (e.g., PD-L1). Small molecule inhibitors include small moleculeantagonists (i.e., small molecules that reduce the effect of anagonist).

As used herein, “specifically binds” and the like refers to the specificassociation or specific binding between the antigen binding domain andthe antigen, as compared with the interaction of the antigen bindingdomain with a different antigen (i.e., non-specific binding). In someembodiments, an antigen binding domain that recognizes or specificallybinds to an antigen has a dissociation constant (KD) of <100 nM, <10 nM,<1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 10⁻⁸ M or less, e.g. from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Specific binding doesnot require that the antigen binding domain does not associate with orbind to any other antigen, but rather that it preferentially associateswith or binds to the antigen, as compared to association with or bindingto an unrelated antigen.

As used herein, “Nectin-4,” also known as poliovirus receptor-relatedprotein 4 (PVRL4), LNIR, PRIM, and EDSS1, is a member of the nectinsubfamily of immunoglobulin-like adhesion molecules that participate inCa²⁺-independent cell-cell adhesion. Nectins bind to the actincytoskeleton through the adaptor protein afadin (AFDN) and are keycomponents of adherens junctions. Nectin-4 contains twoimmunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-typedomain. It may be a single-pass type I membrane protein or a solubleform is produced by proteolytic cleavage at the cell surface by themetalloproteinase ADAM17/TACE. Nectin-4 is overexpressed in multiplehuman cancers, including but not limited to triple negative breastcancer, bladder cancer, urothelial cancer, ovarian cancer, cervicalcancer, endometrial cancer, lung cancer, skin cancer, esophageal cancer,and its abnormal expression may associated with cancer progression andpoor prognosis. Nectin-4 includes mammalian Nectin-4 proteins, e.g.,mouse, rat, rabbit, guinea pig, pig, sheep, dog, non-human primate, andhuman. In some embodiments, Nectin-4 is a human Nectin-4 (encoded byGenBank accession number AF426163) or a mouse Nectin-4 (see Reymond etal., Journal of Biological Chemistry 276:43205-15, 2001).

As used herein, an “immune cell” refers to a T cell, B cell, NK cell,NKT cell, or an antigen presenting cell. In some embodiments, an immunecell is a T cell, B cell, NK cell, or NKT cell. In some embodiments, animmune cell is an antigen presenting cell. In some embodiments, animmune cell is not an antigen presenting cell.

As used herein, an “immune stimulatory compound” is a compound thatactivates or stimulates an immune cell, such as a myeloid cell or anAPC.

As used herein, a “myeloid cell” refers to a dendritic cell, amacrophage, a monocyte, a myeloid derived suppressor cell (MDSC).

As used herein, a “myeloid cell agonist” refers to a compound thatactivates or stimulates an immune response by a myeloid cell.

As used herein, a “benzazepine compound” refers to small moleculechemical compound comprising a benzazepine moiety, where the benzazepinemoiety is a benzene ring fused to a 7-membered ring that comprises oneor two nitrogen ring members. In addition to the bond where the ring isfused to the benzene ring, the 7-membered ring includes two double bonds(e.g., an azepine or diazepine ring), one double bond (e.g., adihydroazepine or dihydro-diazepine ring), or no double bonds (e.g., atetrahydroazepine, azepane, tetrahydrodiazepine, or diazepane ring). Thebenzazepine moiety is optionally substituted. In some embodiments, thebenzazepine moiety is an optionally substituted4,5-dihydro-3H-benzo[b]azepine. In some embodiments, the benzazepinemoiety has the structure:

wherein

is a double bond or a single bond;

L² is selected from —X²—, —X²—C₁₋₆alkylene-X²—, —X²—C₂₋₆ alkenylene-X²—,and —X²—C₂₋₆ alkynylene-X²—, each of which is optionally substituted onalkylene, alkenylene or alkynylene with one or more R¹²;

X² at each occurrence is independently selected from a bond, —O—, —S—,—N(R¹⁰)—, —C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R¹⁰)—,—C(O)N(R¹⁰)C(O)—, —C(O)N(R¹⁰)C(O)N(R¹⁰), —N(R¹⁰)C(O)—,—N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)O—, —OC(O)N(R¹⁰)—, —C(NR¹⁰)—,—N(R¹⁰)C(NR¹⁰)—, —C(NR¹⁰)N(R¹⁰)—, —N(R¹⁰)C(NR¹⁰)N(R¹⁰)—, —S(O)₂—,—OS(O)—, —S(O)O—, —S(O), —OS(O)₂—, —S(O)₂O, —N(R¹⁰)S(O)₂—,—S(O)₂N(R¹⁰)—, —N(R¹⁰)S(O)—, —S(O)N(R¹⁰)—, —N(R¹⁰)S(O)₂N(R¹⁰)—, and—N(R¹⁰)S(O)N(R¹⁰)—;

R¹² is independently selected at each occurrence from halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰,—OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀carbocycle and 3- to 10-membered heterocycle; and C₃₋₁₀ carbocycle and3- to 10-membered heterocycle, wherein each C₃₋₁₀ carbocycle and 3- to10-membered heterocycle in R¹² is optionally substituted with one ormore substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl;

R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle in R⁴ is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl; and

R¹⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, —C₁₋₁₀haloalkyl, —O—C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂ carbocycle, 3- to 12-membered heterocycle,and haloalkyl; and

the moiety is optionally substituted at any position.

The terms “salt” or “pharmaceutically acceptable salt” refer to saltsderived from a variety of organic and inorganic counter ions well knownin the art. Pharmaceutically acceptable acid addition salts can beformed with inorganic acids and organic acids. Inorganic acids fromwhich salts can be derived include, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike. Organic acids from which salts can be derived include, forexample, acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, trifluoroacetic acid and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like. Organic bases fromwhich salts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, specifically such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. In some embodiments,the pharmaceutically acceptable base addition salt is chosen fromammonium, potassium, sodium, calcium, and magnesium salts.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas alkyl, alkenyl, or alkynyl is meant to include groups that containfrom x to y carbons in the chain. For example, the term “C₁₋₆alkyl”refers to substituted or unsubstituted saturated hydrocarbon groups,including straight-chain alkyl and branched-chain alkyl groups thatcontain from 1 to 6 carbons. The term —C_(x-y)alkylene- refers to asubstituted or unsubstituted alkylene chain with from x to y carbons inthe alkylene chain. For example —C₁₋₆alkylene- may be selected frommethylene, ethylene, propylene, butylene, pentylene, and hexylene, anyone of which is optionally substituted.

The terms “C_(x-y)alkenyl” and “C_(x-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond, respectively. The term—C_(x-y)alkenylene- refers to a substituted or unsubstituted alkenylenechain with from x to y carbons in the alkenylene chain. For example,—C₂₋₆alkenylene- may be selected from ethenylene, propenylene,butenylene, pentenylene, and hexenylene, any one of which is optionallysubstituted. An alkenylene chain may have one double bond or more thanone double bond in the alkenylene chain. The term —C_(x-y)alkynylene-refers to a substituted or unsubstituted alkynylene chain with from x toy carbons in the alkenylene chain. For example, —C₂₋₆alkenylene- may beselected from ethynylene, propynylene, butynylene, pentynylene, andhexynylene, any one of which is optionally substituted. An alkynylenechain may have one triple bond or more than one triple bond in thealkynylene chain.

“Alkylene” refers to a divalent hydrocarbon chain linking the rest ofthe molecule to a radical group, consisting solely of carbon andhydrogen, containing no unsaturation, and preferably having from one totwelve carbon atoms, for example, methylene, ethylene, propylene,butylene, and the like. The alkylene chain is attached to the rest ofthe molecule through a single bond and to the radical group through asingle bond. The points of attachment of the alkylene chain to the restof the molecule and to the radical group are through the terminalcarbons respectively. In other embodiments, an alkylene comprises one tofive carbon atoms (i.e., C₁-C₅ alkylene). In other embodiments, analkylene comprises one to four carbon atoms (i.e., C₁-C₄ alkylene). Inother embodiments, an alkylene comprises one to three carbon atoms(i.e., C₁-C₃ alkylene). In other embodiments, an alkylene comprises oneto two carbon atoms (i.e., C₁-C₂ alkylene). In other embodiments, analkylene comprises one carbon atom (i.e., C₁ alkylene). In otherembodiments, an alkylene comprises five to eight carbon atoms (i.e.,C₅-C₈ alkylene). In other embodiments, an alkylene comprises two to fivecarbon atoms (i.e., C₂-C₅ alkylene). In other embodiments, an alkylenecomprises three to five carbon atoms (i.e., C₃-C₅ alkylene). Unlessstated otherwise specifically in the specification, an alkylene chain isoptionally substituted by one or more substituents such as thosesubstituents described herein.

“Alkenylene” refers to a divalent hydrocarbon chain linking the rest ofthe molecule to a radical group, consisting solely of carbon andhydrogen, containing at least one carbon-carbon double bond, andpreferably having from two to twelve carbon atoms. The alkenylene chainis attached to the rest of the molecule through a single bond and to theradical group through a single bond. The points of attachment of thealkenylene chain to the rest of the molecule and to the radical groupare through the terminal carbons respectively. In other embodiments, analkenylene comprises two to five carbon atoms (i.e., C₂-C₅ alkenylene).In other embodiments, an alkenylene comprises two to four carbon atoms(i.e., C₂-C₄ alkenylene). In other embodiments, an alkenylene comprisestwo to three carbon atoms (i.e., C₂-C₃ alkenylene). In otherembodiments, an alkenylene comprises two carbon atoms (i.e., C₂alkenylene). In other embodiments, an alkenylene comprises five to eightcarbon atoms (i.e., C₅-C₈ alkenylene). In other embodiments, analkenylene comprises three to five carbon atoms (i.e., C₃-C₅alkenylene). Unless stated otherwise specifically in the specification,an alkenylene chain is optionally substituted by one or moresubstituents such as those substituents described herein.

“Alkynylene” refers to a divalent hydrocarbon chain linking the rest ofthe molecule to a radical group, consisting solely of carbon andhydrogen, containing at least one carbon-carbon triple bond, andpreferably having from two to twelve carbon atoms. The alkynylene chainis attached to the rest of the molecule through a single bond and to theradical group through a single bond. The points of attachment of thealkynylene chain to the rest of the molecule and to the radical groupare through the terminal carbons respectively. In other embodiments, analkynylene comprises two to five carbon atoms (i.e., C₂-C₅ alkynylene).In other embodiments, an alkynylene comprises two to four carbon atoms(i.e., C₂-C₄ alkynylene). In other embodiments, an alkynylene comprisestwo to three carbon atoms (i.e., C₂-C₃ alkynylene). In otherembodiments, an alkynylene comprises two carbon atoms (i.e., C₂alkynylene). In other embodiments, an alkynylene comprises five to eightcarbon atoms (i.e., C₅-C₈alkynylene). In other embodiments, analkynylene comprises three to five carbon atoms (i.e., C₃-C₅alkynylene). Unless stated otherwise specifically in the specification,an alkynylene chain is optionally substituted by one or moresubstituents such as those substituents described herein.

“Heteroalkylene” refers to a divalent hydrocarbon chain including atleast one heteroatom in the chain, containing no unsaturation, andpreferably having from one to twelve carbon atoms and from one to 6heteroatoms, e.g., —O—, —NH—, —S—. The heteroalkylene chain is attachedto the rest of the molecule through a single bond and to the radicalgroup through a single bond. The points of attachment of theheteroalkylene chain to the rest of the molecule and to the radicalgroup are through the terminal atoms of the chain. In other embodiments,a heteroalkylene comprises one to five carbon atoms and from one tothree heteroatoms. In other embodiments, a heteroalkylene comprises oneto four carbon atoms and from one to three heteroatoms. In otherembodiments, a heteroalkylene comprises one to three carbon atoms andfrom one to two heteroatoms. In other embodiments, a heteroalkylenecomprises one to two carbon atoms and from one to two heteroatoms. Inother embodiments, a heteroalkylene comprises one carbon atom and fromone to two heteroatoms. In other embodiments, a heteroalkylene comprisesfive to eight carbon atoms and from one to four heteroatoms. In otherembodiments, a heteroalkylene comprises two to five carbon atoms andfrom one to three heteroatoms. In other embodiments, a heteroalkylenecomprises three to five carbon atoms and from one to three heteroatoms.Unless stated otherwise specifically in the specification, aheteroalkylene chain is optionally substituted by one or moresubstituents such as those substituents described herein.

The term “carbocycle” as used herein refers to a saturated, unsaturatedor aromatic ring in which each atom of the ring is carbon. Carbocycleincludes 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclicrings, and 6- to 12-membered bridged rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated, and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. A bicyclic carbocycle includes anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits. A bicyclic carbocycle includes any combination of ringsizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fusedring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fusedring systems, 5-8 fused ring systems, and 6-8 fused ring systems.Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl,adamantyl, phenyl, indanyl, and naphthyl. The term “unsaturatedcarbocycle” refers to carbocycles with at least one degree ofunsaturation and excluding aromatic carbocycles. Examples of unsaturatedcarbocycles include cyclohexadiene, cyclohexene, and cyclopentene.

The term “heterocycle” as used herein refers to a saturated, unsaturatedor aromatic ring comprising one or more heteroatoms. Exemplaryheteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3-to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and6- to 12-membered bridged rings. A bicyclic heterocycle includes anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits. In an exemplary embodiment, an aromatic ring, e.g.,pyridyl, may be fused to a saturated or unsaturated ring, e.g.,cyclohexane, cyclopentane, morpholine, piperidine or cyclohexene. Abicyclic heterocycle includes any combination of ring sizes such as 4-5fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8fused ring systems, and 6-8 fused ring systems. The term “unsaturatedheterocycle” refers to heterocycles with at least one degree ofunsaturation and excluding aromatic heterocycles. Examples ofunsaturated heterocycles include dihydropyrrole, dihydrofuran,oxazoline, pyrazoline, and dihydropyridine.

The term “heteroaryl” includes aromatic single ring structures,preferably 5- to 7-membered rings, more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother rings can be aromatic or non-aromatic carbocyclic, orheterocyclic. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons or substitutable heteroatoms, e.g.,—NH—, of the structure. It will be understood that “substitution” or“substituted with” includes the implicit proviso that such substitutionis in accordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,i.e., a compound which does not spontaneously undergo transformationsuch as by rearrangement, cyclization, elimination, etc. In certainembodiments, substituted refers to moieties having substituentsreplacing two hydrogen atoms on the same carbon atom, such assubstituting the two hydrogen atoms on a single carbon with an oxo,imino or thioxo group. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of thisdisclosure, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.

In some embodiments, substituents may include any substituents describedherein, for example: halogen, hydroxy, oxo (═O), thioxo (═S), cyano(—CN), nitro (—NO₂), imino (═N—H), oximo (═N—OH), hydrazino (═N—NH₂),—R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a),—R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2), and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); and alkyl, alkenyl,alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl,and heteroarylalkyl any of which may be optionally substituted by alkyl,alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo(═O), thioxo (═S), cyano (—CN), nitro (—NO₂), imino (═N—H), oximo(═N—OH), hydrazine (═N—NH₂), —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a),—R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂,—R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂,—R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a),—R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2);wherein each R^(a) is independently selected from hydrogen, alkyl,cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein eachR^(a), valence permitting, may be optionally substituted with alkyl,alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo(═O), thioxo (═S), cyano (—CN), nitro (—NO₂), imino (═N—H), oximo(═N—OH), hydrazine (═N—NH₂), —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a),—R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂,—R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂,—R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a),—R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2);and wherein each R^(b) is independently selected from a direct bond or astraight or branched alkylene, alkenylene, or alkynylene chain, and eachR^(c) is a straight or branched alkylene, alkenylene or alkynylenechain.

It will be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to a“heteroaryl” group or moiety implicitly includes both substituted andunsubstituted variants.

In addition, it should be understood that the individual compounds(e.g., proteins), or groups of compounds, derived from the variouscombinations of the structures and substituents (e.g., domains, regionsor peptide components) described herein, are disclosed by the presentapplication to the same extent as if each compound or group of compoundswas set forth individually. Thus, selection of particular structures orparticular substituents is within the scope of the present disclosure.

Chemical entities having carbon-carbon double bonds or carbon-nitrogendouble bonds may exist in Z- or E-form (or cis- or trams-form).Furthermore, some chemical entities may exist in various tautomericforms. Unless otherwise specified, chemical entities described hereinare intended to include all Z-, E- and tautomeric forms as well.

A “tautomer” refers to a molecule wherein a proton shift from one atomof a molecule to another atom of the same molecule is possible. Thecompounds presented herein, in certain embodiments, exist as tautomers.In circumstances where tautomerization is possible, a chemicalequilibrium of the tautomers will exist. The exact ratio of thetautomers depends on several factors, including physical state,temperature, solvent, and pH. Some examples of tautomeric equilibriuminclude:

As used herein, a “conjugate” refers to an antibody or antigen bindingfragment thereof attached to at least one immune stimulatory compound,optionally via a linker.

The phrases “intravenous administration” and “administeredintravenously” as used herein refer to injection or infusion of aconjugate into a vein of a subject.

The phrases “intravenous slow infusion” and “IV slow infusion” as usedhere refer to an intravenous infusion that results in a T max of about 4hours or more.

The phrases “subcutaneous administration”, “subcutaneouslyadministering” and the like refer to administration of a conjugate intothe subcutis of a subject. For clarity, a subcutaneous administration isdistinct from an intratumoral injection into a tumor or cancerous lesionlocated in the subcuta.

The phrase “pharmaceutically acceptable” refers to compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Anti-Nectin-4 Antibodies

In one aspect, antibodies (e.g., isolated monoclonal antibodies) thatspecifically bind to Nectin-4, also referred to as anti-Nectin-4antibodies, or antigen-binding fragments thereof, are provided.

In various embodiments, an antibody or antigen binding fragment thereofcomprises two light chain polypeptides (light chains) and two heavychain polypeptides (heavy chains), held together covalently by disulfidelinkages.

The heavy chain typically comprises a heavy chain variable region (VH)and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2, and CH3. Nonlimiting exemplary heavychain constant regions include human IgG1, human IgG2, human IgG3, andhuman IgG4 constant regions. In some embodiments, an antibody providedherein comprises an IgG1 constant region.

The light chain typically comprises a light chain variable region (VL)and a light chain constant region. Nonlimiting exemplary light chainconstant regions include kappa and lambda constant regions. Anonlimiting exemplary human kappa constant region is shown in SEQ IDNO:20. Another exemplary light chain constant region is mouse kappaconstant region shown in SEQ ID NO:22.

The constant domains provide the general framework of the antibody andmay not be involved directly in binding the antibody to an antigen, butcan be involved in various effector functions, such as participation ofthe antibody in antibody-dependent cellular cytotoxicity (ADCC), ADCP(antibody-dependent cellular phagocytosis), CDC (complement-dependentcytotoxicity) and complement fixation, binding to Fc receptors (e.g.,CD16, CD32, FcRn), greater in vivo half-life relative to a polypeptidelacking an Fc region, protein A binding, and perhaps even placentaltransfer (see Capon et al., Nature 357:525, 1989). As used herein, “anFc region constant domain portion” or “Fc region portion” refers to theheavy chain constant region segment of the Fc fragment (the “fragmentcrystallizable” region or Fc region) from an antibody, which can ininclude one or more constant domains, such as CH2, CH3, CH4, or anycombination thereof. In certain embodiments, an Fc region portionincludes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody and anycombination thereof, or the CH3 and CH4 domains of an IgM or IgEantibody and any combination thereof.

An Fc region or domain may interact with different types of FcRs. Thedifferent types of FcRs may include, for example, FcγRI, FcγRIIA,FcγRIIB, FcγRIIIA, FcγRIIIB, FcαRI, FcμR, FcεRI, FcεRII, and FcRn. FcRsmay be located on the membrane of certain immune cells including, forexample, B lymphocytes, natural killer cells, macrophages, neutrophils,follicular dendritic cells, eosinophils, basophils, platelets, and mastcells. Once the FcR is engaged by the Fc domain, the FcR may initiatefunctions including, for example, clearance of an antigen-antibodycomplex via receptor-mediated endocytosis, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody dependent cell-mediatedphagocytosis (ADCP), trogocytosis, trogoptosis, and ligand-triggeredtransmission of signals across the plasma membrane that can result inalterations in secretion, exocytosis, and cellular metabolism. FcRs maydeliver signals when FcRs are aggregated by antibodies and multivalentantigens at the cell surface. The aggregation of FcRs withimmunoreceptor tyrosine-based activation motifs (ITAMs) may sequentiallyactivate SRC family tyrosine kinases and SYK family tyrosine kinases.ITAM comprises a twice-repeated YxxL sequence flanking seven variableresidues. The SRC and SYK kinases may connect the transduced signalswith common activation pathways.

In some embodiments, an Fc region or domain can exhibit reduced bindingaffinity to one or more Fc receptors. In some embodiments, an Fc regionor domain can exhibit reduced binding affinity to one or more Fcγreceptors. In some embodiments, an Fc region or domain can exhibitreduced binding affinity to FcRn receptors. In some embodiments, an Fcregion or domain can exhibit reduced binding affinity to Fcγ and FcRnreceptors. In some embodiments, an Fc domain is an Fc null domain orregion. As used herein, an “Fc null” refers to a domain that exhibitsweak to no binding to any of the Fcgamma receptors. In some embodiments,an Fc null domain or region exhibits a reduction in binding affinity(e.g., increase in Kd) to Fcγ receptors of at least about 1000-fold.

The Fc region or domain may have one or more, two or more, three ormore, or four or more, or up to five amino acid substitutions thatdecrease binding of the Fc region or domain to an Fc receptor. In someembodiments, an Fc region or domain exhibits decreased binding to FcγRI(CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or anycombination thereof. In order to decrease binding affinity of an Fcregion or domain to an Fc receptor, an Fc region or domain may compriseone or more amino acid substitutions that has the effect of reducing theaffinity of the Fc domain or region to an Fc receptor. In certainembodiments, the Fc region or domain is an IgG1 and the one or moresubstitutions in the Fc region or domain comprise any one or more ofIgG1 heavy chain mutations corresponding to E233P, L234V, L234A, L235A,L235E, ΔG236, G237A, E318A, K320A, K322A, A327G, A330S, or P331Saccording to the EU index of Kabat numbering.

In some embodiments, the Fc region or domain can comprise a sequence ofthe IgG1 isoform that has been modified from the wild-type IgG1sequence. A modification can comprise a substitution at more than oneamino acid residue, such as at 5 different amino acid residues includingL235V/F243L/R292P/Y300L/P396L (IgG1VLPLL) according to the EU index ofKabat numbering. A modification can comprise a substitution at more thanone amino acid residues, such as at 2 different amino acid residuesincluding S239D/I332E (IgG1DE) according to the EU index of Kabatnumbering. A modification can comprise a substitution at more than oneamino acid residue, such as at 3 different amino acid residues includingS298A/E333A/K334A (IgG1AAA) according to the EU index of Kabatnumbering. Non-limiting exemplary IgG1 constant regions are shown in SEQID NOs: 18 and 19. In certain other embodiments, an antibody providedherein comprises a mouse IgG2a heavy chain constant region shown in SEQID NO:21.

An antibody or Fc domain may be modified to acquire or improve at leastone constant region-mediated biological effector function relative to anunmodified antibody or Fc domain, e.g., to enhance FcγR interactions. Incertain embodiments, a modification can increase CD32b binding (andsupport transdelivery in a PBMC assay) comprises a substitution at S267Land E329F (IgG1LF, also known as SELF double mutant) according to the EUindex of Kabat numbering. For example, an antibody with a constantregion that binds to FcγRIIA, FcγRIIB and/or FcγRIIIA with greateraffinity than the corresponding wild type constant region may beproduced according to the methods described herein. An Fc domain thatbinds to FcγRIIA, FcγRIIB and/or FcγRIIIA with greater affinity than thecorresponding wild type Fc domain may be produced according to themethods described herein.

In certain embodiments, an Fc region or domain found in an anti-Nectin-4antibody of the present disclosure will be capable of mediating one ormore of these effector functions, or will lack one or more or all ofthese activities or have one or more of the effector activitiesincreased by way of, for example, one or more mutations as compared tothe unmodified Fc region or domain.

The antigen-recognition regions of the antibody variable domainstypically comprise six complementarity determining regions (CDRs), orhypervariable regions, that lie within the framework of the heavy chainvariable region and light chain variable region at the N-terminal endsof the two heavy and two light chains.

In some embodiments, an antigen binding domain comprises a light chaincomplementary determining region 1 (LCDR1), a light chain complementarydetermining region 2 (LCDR2), a light chain complementary determiningregion 3 (LCDR3), a heavy chain complementary determining region 1(HCDR1), a heavy chain complementary determining region 2 (HCDR2), and aheavy chain complementary determining region 3 (HCDR3). In someembodiments, an antibody may be a heavy-chain only antibody, in whichcase the antigen binding domain comprises HCDR1, HCDR2, and HCDR3, andthe antibody lacks a light chain.

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises a heavy chain variable region (VH) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ IDNO:9, and a light chain variable region (VL) comprising an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO: 11, providedthat the amino acid sequences of the VH-CDRs (i.e., SEQ ID NOS:1-3) andVL-CDRs (i.e., SEQ ID NOS:4, 7, and 8) are unchanged. In someembodiments, the antibody or antigen binding fragment thereof comprisesa heavy chain comprising an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence selected from SEQ ID NO:23, and a light chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ IDNO:25, provided that the amino acid sequences of the VH-CDRs and VL-CDRsare unchanged.

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence selected from SEQ ID NOS:4-6, a VL-CDR2 comprising the aminoacid sequence of SEQ ID NO:7, and a VL-CDR3 comprising the amino acidsequence comprising SEQ ID NO:8. In some such embodiments, the antibodyor antigen binding fragment thereof comprises a heavy chain variableregion (VH) comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 10, and a light chain variable region (VL)comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequenceselected from SEQ ID NOS: 12-17, provided that the amino acid sequencesof the VH-CDRs and VL-CDRs are unchanged. In some embodiments, theantibody or antigen binding fragment thereof comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO:24, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence selected from SEQ ID NOS:26-31,provided that the amino acid sequences of the VH-CDRs and VL-CDRs areunchanged.

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising an amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising an amino acid sequence of SEQ ID NO: 12,or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In certain embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:13, or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:5, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:14, or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:6, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:15, or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:6, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:16, or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: a heavy chain CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:5, a VL-CDR2 comprising the amino acid sequence ofSEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence comprisingSEQ ID NO:8. In some such embodiments, the antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 10, (b) a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:17, or both (a) and (b).

In some embodiments, an anti-Nectin-4 antibody or antigen bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO:9, and a light chainvariable region (VL) comprising the amino acid of SEQ ID NO: 11; or (b)a heavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 10, and a light chain variable region (VL) comprising theamino acid sequence selected from SEQ ID NOs:12-17.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 12.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 13.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 14.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 15.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 16.

In some such embodiments, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 10 and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, an anti-Nectin-4 antibody comprises: (a) a heavychain comprising the amino acid sequence of SEQ ID NO:23, and a lightchain comprising the amino acid sequence of SEQ ID NO:25; or (b) a heavychain comprising the amino acid sequence of SEQ ID NO:24, and a lightchain comprising the amino acid sequence selected from SEQ ID NOS:26-31.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:26.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:27.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:28.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:29.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:30.

In some such embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:31.

In any of the aforementioned embodiments, the anti-Nectin-4 antibody orantigen binding fragment thereof is conjugated to a small molecule drugto form an antibody drug conjugate. In certain embodiments, the smallmolecule drug is a myeloid cell agonist (e.g., TLR8 agonist) asdisclosed herein, thus forming a myeloid cell agonist conjugate.

An anti-Nectin-4 antibody or antigen binding fragment thereof can bechimeric or humanized. Chimeric and humanized forms of non-human (e.g.,murine) antibodies can be intact (full length) chimeric immunoglobulins,immunoglobulin chains or antigen binding fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subdomains of antibodies),which can contain sequences derived from non-human immunoglobulin. Ingeneral, the humanized antibody or antigen binding fragment thereof cancomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin sequence.A humanized antibody can also comprise at least a portion of animmunoglobulin constant region (Fc), an Fc domain, typically that of ahuman immunoglobulin sequence.

An anti-Nectin-4 antibody or antigen binding fragment thereof describedherein can be a human antibody. As used herein, “human antibodies” caninclude antibodies having, for example, the amino acid sequence of ahuman immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulins and that typically do not express endogenousimmunoglobulins. Human antibodies can be produced using transgenic miceincapable of expressing functional endogenous immunoglobulins, butcapable of expressing human immunoglobulin genes. Completely humanantibodies that recognize a selected epitope can be generated usingguided selection. In this approach, a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope.

An anti-Nectin-4 antibody or antigen binding fragment thereof describedherein can be a bispecific antibody or a dual variable domain antibody(DVD). Bispecific and DVD antibodies are monoclonal, often human orhumanized, antibodies that have binding specificities for at least twodifferent antigens, one of which is Nectin-4.

An anti-Nectin-4 antibody or antigen binding fragment thereof describedherein can be derivatized or otherwise modified. For example,derivatized antibodies can be modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or the like.

Antibody CDRs

Nectin-4 antibody CDR sequences may be determined by one or moremethods, including Kabat, Chothia, AbM, Contact, IMGT and AHo (see TableA below). Unless otherwise specified herein, CDR sequences aredetermined according to the Kabat method. References to variable regionor CDR numbering as in Kabat, amino acid position numbering as in Kabat,or CDR sequences determined according to the Kabat method, andvariations thereof, refer to the numbering system used for heavy chainvariable regions or light chain variable regions of the compilation ofantibodies in Kabat et al. ((1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). Using this numberingsystem, the actual linear amino acid sequence may contain fewer oradditional amino acids corresponding to a shortening of, or insertioninto, an FR or CDR of the variable domain. For example, a heavy chainvariable domain may include a single amino acid insert (residue 52aaccording to Kabat) after residue 52 and three inserted residues (e.g.,residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.The Kabat numbering of residues may be determined for a given antibodyby alignment at regions of homology of the sequence of the antibody witha “standard” Kabat numbered sequence. The Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately residues 1-107 of the light chain and residues 1-113 ofthe heavy chain) (e.g., Kabat et al., supra).

The “EU numbering system” or “EU index” is generally used when referringto a residue in an immunoglobulin heavy chain constant region (e.g., theEU index reported in Kabat et al., supra). The “EU index, as in Kabat,”refers to the residue numbering of the human IgG 1 EU antibody.

Other numbering systems have been described, for example, by AbM (OxfordMolecular's AbM antibody modeling software (see, e.g., AntibodyEngineering Vol. 2 (Kontermann and Dithel eds., 2d ed. 2010)), Chothia(see, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17), Contact, IMGT(ImMunoGeneTics (IMGT) Information System® (see, Lafranc et al., 2003,Dev. Comp. Immunol. 27(1):55-77)), and AHon (see, Honegger andPlückthun, 2001, J. Mol. Biol. 309: 657-70) and are well understood by aperson of ordinary skill in the art.

In certain embodiments, an anti-Nectin-4 antibody of this disclosure iscomprised of (a) a heavy chain variable region (VH) comprising a CDR1(VH-CDR1) comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2comprising the amino acid sequence of SEQ ID NO:2, and a VH-CDR3comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chainvariable region (VL) comprising a CDR1 (VL-CDR1) comprising the aminoacid sequence selected from any one of SEQ ID NOS:4-6, a VL-CDR2comprising the amino acid sequence of SEQ ID NO:7, and a VL-CDR3comprising the amino acid sequence of SEQ ID NO:8.

In further embodiments, an anti-Nectin-4 antibody of this disclosure iscomprised of (a) a VH comprising a CDR1 (VH-CDR1) comprising the aminoacid sequence of SEQ ID NO:33, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:35; and (b) a VL comprising a CDR1 (VL-CDR1)comprising the amino acid sequence of SEQ ID NO:36, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:37, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:38.

In still further embodiments, an anti-Nectin-4 antibody of thisdisclosure is comprised of (a) a VH comprising a CDR1 (VH-CDR1)comprising the amino acid sequence of SEQ ID NO:39, a VH-CDR2 comprisingthe amino acid sequence of SEQ ID NO:40, and a VH-CDR3 comprising theamino acid sequence of SEQ ID NO:41; and (b) a VL comprising a CDR1(VL-CDR1) comprising the amino acid sequence of SEQ ID NO:42, a VL-CDR2comprising the amino acid sequence of SEQ ID NO:43, and a VL-CDR3comprising the amino acid sequence of SEQ ID NO:44.

In yet further embodiments, an anti-Nectin-4 antibody of this disclosureis comprised of (a) a VH comprising a CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:45, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:46, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:47; and (b) a VL comprising a CDR1 (VL-CDR1)comprising the amino acid sequence of SEQ ID NO:48, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:49, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:50.

In yet further embodiments, an anti-Nectin-4 antibody of this disclosureis comprised of (a) a VH comprising a CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:51, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:52, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:53; and (b) a VL comprising a CDR1 (VL-CDR1)comprising the amino acid sequence of SEQ ID NO:54, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:55, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:56.

In yet further embodiments, an anti-Nectin-4 antibody of this disclosureis comprised of (a) a VH comprising a CDR1 (VH-CDR1) comprising theamino acid sequence of SEQ ID NO:57, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:58, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:59; and (b) a VL comprising a CDR1 (VL-CDR1)comprising the amino acid sequence of SEQ ID NO:60, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:61, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:62.

TABLE A Anti-Nectin4 CDRs Antibody CDR Identity Kabat Chothia AbmContact IMGT AHo D6C VH NYDMS GFTFSNY (SEQ GFTFSNYDMS SNYDMS GFTFSNYDSGFTFSNYDM CDR1 (SEQ ID NO: 1) ID NO: 33) (SEQ ID NO: 39) (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 57) 45) 51) D6C VH TISSGGSYTY SSGGSY (SEQTISSGGSYTY WVATISSGG ISSGGSYT SSGGSYTYYVDS CDR2 YVDSVKG ID NO: 34)(SEQ ID NO: 40) SYTY (SEQ (SEQ ID NO: VKGRF (SEQ ID (SEQ ID NO: 2)ID NO: 46) 52) NO: 58) D6C VH QELGSYYAM QELGSYYAMD QELGSYYAMD ARQELGSYYARQELGSYY ELGSYYAMDY CDR3 DY Y (SEQ ID NO: Y (SEQ ID NO: AMD (SEQ IDAMDY (SEQ (SEQ ID NO: 59) (SEQ ID NO: 3)  35) 41) NO: 47) ID NO: 53)D6.2C RSSQSIVHSN RSSQSIVHSNG RSSQSIVHSNG VHSNGNTYL QSIVHSNGNTSSQSIVHSNGNT and ANTYLE NTYLE (SEQ ID NTYLE (SEQ ID EWY (SEQ IDY (SEQ ID NO:  Y (SEQ ID NO: 60) D6.5C (SEQ ID NO: 5)  NO: 36) NO: 42)NO: 48) 54) VL CDR1 D6C VL KVSNRFS KVSNRFS (SEQ KVSNRFS (SEQ LLIYKVSNRKVS (SEQ ID KVSNRFSGVPDR CDR2 (SEQ ID NO: 7)  ID NO: 37) ID NO: 43)F (SEQ ID NO: 55) (SEQ ID NO: 61) NO: 49) D6C VL FQGSHVPYT FQGSHVPYTFQGSHVPYT FQGSHVPY FQGSHVPYT GSHVPYTF (SEQ CDR3 (SEQ ID NO: 8)(SEQ ID NO: 38) (SEQ ID NO: 44) (SEQ ID NO: (SEQ ID ID NO: 62) 50)NO: 56)Nucleic Acids. Vectors, and Host Cells

The present disclosure provides an isolated nucleic acid that encodesanti-Nectin-4 antibody or antigen binding fragment thereof as describedherein. In some embodiments, the nucleic acid encoding the anti-Nectin-4antibody or antigen binding fragment thereof is codon optimized toenhance or maximize expression in certain types of cells (e.g., Scholtenet al., Clin. Immunol. 119: 135-145, 2006). As used herein a “codonoptimized” polynucleotide is a heterologous polypeptide having codonsmodified with silent mutations corresponding to the abundances of hostcell tRNA levels.

In some embodiments, a nucleic acid molecule encodes an anti-Nectin-4antibody or antigen binding fragment thereof (e.g., an antibody heavyand light chains, or an antibody binding domain comprising VH and VLbinding regions) as disclosed herein wherein two or more chains orregions are separated by a cleavage site. In some embodiments, thecleavage site is a self-cleaving amino acid sequence comprising a 2Apeptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A),Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or anycombination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011,which 2A nucleic acid and amino acid sequences are incorporated hereinby reference in their entirety).

In another aspect, an expression construct comprising a nucleic acidencoding an anti-Nectin-4 antibody or antigen binding fragment thereofas described herein is provided. In some embodiments, a nucleic acid maybe operably linked to an expression control sequence. As used herein,“expression construct” refers to a DNA construct containing a nucleicacid molecule that is operably-linked to a suitable control sequencecapable of effecting the expression of the nucleic acid molecule in asuitable host. An expression construct may be present in a vector (e.g.,a bacterial vector, a viral vector) or may be integrated into a genome.The term “operably linked” refers to the association of two or morenucleic acids on a single polynucleotide fragment so that the functionof one is affected by the other. For example, a promoter isoperably-linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., the coding sequence isunder the transcriptional control of the promoter). The term “expressioncontrol sequence” (also called a regulatory sequence) refers to nucleicacid sequences that effect the expression and processing of codingsequences to which they are operably linked. For example, expressioncontrol sequences may include transcription initiation, termination,promoter and enhancer sequences; efficient RNA processing signals suchas splicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequences); sequences that enhance protein stability;and possibly sequences that enhance protein secretion.

In some embodiments, a nucleic acid or an expression construct encodingan anti-Nectin-4 antibody or antigen binding fragment thereof is presentin a vector. A “vector” is a nucleic acid molecule that is capable oftransporting another nucleic acid. Vectors may be, for example,plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA orRNA molecule that may include chromosomal, non-chromosomal,semi-synthetic or synthetic nucleic acids. Exemplary vectors are thosecapable of autonomous replication (episomal vector) or expression ofnucleic acids to which they are linked (expression vectors). Exemplaryviral vectors include retrovirus, adenovirus, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabiesand vesicular stomatitis virus), paramyxovirus (e.g., measles andSendai), positive strand RNA viruses such as picomavirus and alphavirus,and double-stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996). In some embodiments, a vector is aplasmid. In some other embodiments, a vector is a viral vector. In somesuch embodiments, the viral vector is a lentiviral vector or aγ-retroviral vector.

In yet another aspect, the disclosure provides an isolated host cellcomprising a nucleic acid, expression construct, or vector encoding ananti-Nectin-4 antibody or antigen binding fragment thereof, as describedherein. As used herein, the term “host” refers to a cell ormicroorganism targeted for genetic modification with a heterologous orexogenous nucleic acid molecule to produce a polypeptide of interest(e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof).In certain embodiments, a host cell may optionally already possess or bemodified to include other genetic modifications that confer desiredproperties related or unrelated to biosynthesis of the heterologous orexogenous protein (e.g., inclusion of a detectable marker). More thanone heterologous or exogenous nucleic acid molecule can be introducedinto a host cell as separate nucleic acid molecules, as a plurality ofindividually controlled genes, as a polycistronic nucleic acid molecule,as a single nucleic acid molecule encoding a fusion protein, or anycombination thereof. When two or more exogenous nucleic acid moleculesare introduced into a host cell, it is understood that the two moreexogenous nucleic acid molecules can be introduced as a single nucleicacid molecule (e.g., on a single vector), on separate vectors,integrated into the host chromosome at a single site or multiple sites.The number of referenced heterologous nucleic acid molecules or proteinactivities refers to the number of encoding nucleic acid molecules orthe number of protein activities, not the number of separate nucleicacid molecules introduced into a host cell.

Methods for Producing Ant-Nectin-4 Antibodies

Anti-Nectin-4 antibodies or antigen binding fragments thereof of thisdisclosure can be produced by any method known in the art for antibodyproduction. As one example, an anti-Nectin-4 antibody or antigen bindingfragment can be produced by a method using an isolated nucleic acidsequence encoding an anti-Nectin-4 antibody or antigen binding fragmentthereof, vectors and host cells comprising the nucleic acid sequence,and recombinant techniques for the production of the antibody or antigenbinding fragment thereof. The nucleic acid sequence encoding theanti-Nectin-4 antibody or antigen binding fragment thereof can beisolated into a replicable DNA vector for further cloning or forexpression. DNA encoding an anti-Nectin-4 antibody or antigen bindingfragment thereof can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors known in the art can be used as avector. The vector components generally can include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription-termination sequence.

Suitable host cells for cloning or expressing the DNA vectors herein canbe prokaryote, yeast, or higher eukaryote cells described herein.Suitable host cells for expression of glycosylated antibody or antigenbinding fragment can be derived from multicellular organisms. Examplesof invertebrate cells can include, but are not limited to, plant andinsect cells. Host cells used to produce an antibody or antigen bindingfragment can be cultured in a variety of commercial media. When usingrecombinant techniques, an antibody or antigen binding fragment can beproduced, for example, intracellularly, in the periplasmic space, ordirectly secreted into the medium. If the antibody or antigen bindingfragment is produced intracellularly, the particulate debris, eitherhost cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Where the antibody or antigen bindingfragment is secreted into the medium, supernatants from such expressionsystems can be concentrated using a commercially available proteinconcentration filter. A protease inhibitor such as phenylmethylsulphonylfluoride can be included in any of the foregoing steps to inhibitproteolysis, and antibiotics can be included to prevent the growth ofadventitious contaminants.

An anti-Nectin-4 antibody or antigen binding fragment thereofcomposition prepared from the cells can be purified using, for example,hydroxylapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography. The suitability of a protein A as an affinityligand can depend on the species and isotype of any immunoglobulin Fcdomain that may be present in the antibody or antigen binding fragment.Other techniques for protein purification such as fractionation on thean ion-exchange column, ethanol precipitation, reverse-phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™,chromatography on an anion- or cation-exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, andammonium-sulfate precipitation can also be used to recover the antibodyor antigen binding fragment. Following any preliminary purificationstep(s), the mixture comprising the antibody or antigen binding fragmentand contaminants can be subjected to low-pH hydrophobic-interactionchromatography. The methods for humanizing antibodies can include, forexample, humanization uses CDR grafting (Jones et al., Nature 15 321:522(1986)) and variants thereof, including “reshaping” (Verhoeyen, et al.,1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337;Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization”(Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al.,1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol148:1149-1154), and “veneering” (Mark, et al., B W Metcalf, B J Dalton(Eds.) Cellular adhesion: molecular definition to therapeutic potential.Plenum Press, New York; 1994:291-312). Superhumanization (Tan, et al.,2002 J Immunol 169: 1119-25) is another variant humanization method thatcan be used to graft non-human CDRs into human germline antibodysequences having similar CDR canonical structures.

In certain embodiments, the anti-Nectin-4 antibodies or antigen bindingfragments thereof of this disclosure, or conjugates thereof having amyeloid cell agonist, are humanized.

Immune-Stimulatory Compounds

Anti-Nectin-4 antibodies or antigen binding fragments thereof of thisdisclosure are attached to immune stimulatory compounds (e.g., TLR8agonist), generally via a linker(s) to form immune-stimulatoryconjugates. An anti-Nectin-4 antibody or antigen binding fragmentthereof of this disclosure can be attached to one or moreimmune-stimulatory compounds, generally from about 1 to about 10compounds per antibody or antigen binding fragment thereof, andpreferably from about 2 to about 4 compounds per antibody or antigenbinding fragment thereof.

In some embodiments, an immune stimulatory compound activates humanimmune cells, such as dendritic cells, macrophages, monocytes,myeloid-derived suppressor cells, NK cells, B cells, T cells, or acombination thereof. In some embodiments, an immune-stimulatory compoundis a myeloid cell agonist. A myeloid cell agonist is a compound thatactivates or stimulates an immune response by a myeloid cell. Forexample, a myeloid cell agonist can stimulate an immune response bycausing the release of cytokines by myeloid cells, which results in theactivation of immune cells. The stimulation of an immune response by amyeloid cell agonist can be measured in vitro by co-culturing immunecells (e.g., peripheral blood mononuclear cells (PBMCs)) with cellstargeted by the conjugate and measuring cytokine release, chemokinerelease, proliferation of immune cells, upregulation of immune cellactivation markers, ADCP, and/or ADCC. Exemplary assays are described inthe Examples. ADCC can be measured by determining the percentage ofremaining target cells in the co-culture after administration of theconjugate with the target cells and PBMCs.

For example, an immune stimulatory compound can act on toll likereceptors (TLRs), nucleotide-oligomerization domain-like receptors(NOD), RIG-I-Like receptors (RLR), c-type lectin receptors (CLR), orcytosolic DNA Sensors (CDS), or a combination thereof.

In some embodiments, an immune stimulatory compound comprises a ligandof one or more TLRs selected from the group consisting of: TLR2, TLR3,TLR4, TLR5, TLR7, TLR8, TLR9, and TLR10.

In some embodiments, an immune-stimulatory compound is a myeloid cellagonist. In some embodiments, a myeloid cell agonist is a ligand of TLR2selected from the group consisting of: (a) a heat killed bacteriaproduct, preferably HKAL, HKEB, HKHP, HKLM, HKLP, HKLR, HKMF, HKPA,HKPG, or HKSA, HKSP, and (b) a cell-wall components product, preferablyLAM, LM, LPS, LIA, LIA, PGN, FSL, Pam2CSK4, Pam3CSK4, or Zymosan.

In some embodiments, a myeloid cell agonist is a ligand of TLR3 selectedfrom the group consisting of: rintatolimod, poly-ICLC, RIBOXXON®,Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1.

In some embodiments, a myeloid cell agonist is a ligand of TLR4 selectedfrom the group consisting of LPS, MPLA or a pyrimido[5,4-b]indole suchas those described in International Publication No. WO 2014/052828(Regents of the University of California).

In some embodiments, the myeloid cell agonist is a ligand of TLR5selected from the group consisting of: FLA and Flagellin.

In some embodiments, the myeloid cell agonist is a ligand of TLR6.

In certain embodiments, a myeloid cell agonist is a TLR7 agonist and/ora TLR8 agonist. In certain embodiments, the myeloid cell agonist is aTLR7 agonist. In certain embodiments, the myeloid cell agonist is a TLR8agonist. In some embodiments, the myeloid cell agonist selectivelyagonizes TLR7 and not TLR8. In other embodiments, the myeloid cellagonist selectively agonizes TLR8 and not TLR7.

In certain embodiments, a myeloid cell agonist is a TLR7 agonist. Incertain embodiments, the TLR7 agonist is selected from animidazoquinoline, an imidazoquinoline amine, a thiazoquinoline, anaminoquinoline, an aminoquinazoline, apyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a2-aminoimidazole, an 1-alkyl-1H-benzimidazol-2-amine, atetrahydropyridopyrimidine, a heteroarothiadiazide-2,2-dioxide, abenzonaphthyridine, a thieno[3,2-d]pyrimidine, a4-amino-imidazoquinoline, an imidazo-pyridinone, animidazo-pyrimidinone, a purine, a fused pyrimidine-lactam, animidazo[4,5-c]quinoline-4-amine, an imidazo[4,5-c]quinoline, apyrimidine, a benzazepine, an imidazo-pyridine, a pyrrolo-pyrimidine, a2-amino-quinazoline, a guanosine analog, an adenosine analog, athymidine homopolymer, an ssRNA, CpG-A, PolyG10, and PolyG3. In certainembodiments, the TLR7 agonist is selected from an imidazoquinoline, animidazoquinoline amine, a thiazoquinoline, an aminoquinoline, anaminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, apyrimidine-2,4-diamine, a 2-aminoimidazole, a1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, aheteroarothiadiazide-2,2-dioxide, a benzonaphthyridine, athieno[3,2-d]pyrimidine, a 4-amino-imidazoquinoline, animidazo-pyridinone, an imidazo-pyrimidinone, a purine, a fusedpyrimidine-lactam, an imidazo[4,5-c]quinoline-4-amine, animidazo[4,5-c]quinoline, a pyrimidine, a benzazepine, animidazo-pyridine, a pyrrolo-pyrimidine, and a 2-amino-quinazoline, butis other than a guanosine analog, an adenosine analog, a thymidinehomopolymer, an ssRNA, CpG-A, PolyG10, and PolyG3. In some embodiments,a TLR7 agonist is a non-naturally occurring compound. Examples of TLR7modulators include GS-9620, GSK-2245035, imiquimod, resiquimod,DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922,3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7795, and the TLR7modulator compounds disclosed in US Patent Application Publication No.US 2016/0168164 (Janssen, thieno[3,2-d]pyrimidine derivatives), USPatent Application Publication No. US 2015/0299194 (Roche,4-amino-imidazoquinoline derivatives), US Patent Application PublicationNo. US 2011/0098248 (Gilead Sciences, imidazo-pyridinone,imidazo-pyrimidinone, and purine derivatives), US Patent ApplicationPublication No. US 2010/0143301 (Gilead Sciences, fusedpyrimidine-lactam derivatives), and US Patent Application PublicationNo. US 20090047249 (Gilead Sciences, purine derivatives), and thesepublications are incorporated by reference herein. Further examples ofTLR7 modulators include compounds disclosed in International PublicationNo. WO 2018/009916 (Stanford University/Bolt Biotherapeutics,imidazo[4,5-c]quinolin-4-amine derivatives), International PublicationNo. WO 2018/112108 (Bolt Biotherapeutics, imidazo[4,5-c]quinoline,pyrimidine, benzazepine, imidazo-pyridine, pyrrolo-pyrimidine, andpurine derivatives), US Patent Application Publication No. US2019/0055247 (Bristol-Myers Squibb, purine derivatives), InternationalPublication No. WO 2018/198091 (Novartis, pyrrolo-pyrimidinederivatives), US Patent Application Publication No. US 2017/0121421(Novartis, pyrrolo-pyrimidine derivatives), U.S. Pat. No. 10,253,003(Janssen, 2-amino-quinazoline derivatives), and U.S. Pat. No. 10,233,184(Roche, imidazo-pyrimidinone derivatives), and these publications areincorporated by reference herein. In some embodiments, a TLR7 agonisthas an EC50 value of 500 nM or less by PBMC assay measuring TNFalpha orIFNalpha production. In some embodiments, a TLR7 agonist has an EC50value of 100 nM or less by PBMC assay measuring TNFalpha or IFNalphaproduction. In some embodiments, a TLR7 agonist has an EC50 value of 50nM or less by PBMC assay measuring TNFalpha or IFNalpha production. Insome embodiments, a TLR7 agonist has an EC50 value of 10 nM or less byPBMC assay measuring TNFalpha or IFNalpha production.

In certain embodiments, the myeloid cell agonist is a TLR8 agonist. Incertain embodiments, a TLR8 agonist is selected from the groupconsisting of a benzazepine, an imidazoquinoline, a thiazoloquinoline,an aminoquinoline, an aminoquinazoline, apyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a2-aminoimidazole, an 1-alkyl-1H-benzimidazol-2-amine, atetrahydropyridopyrimidine, a pyrido[3,2-d]pyrimidine, adihydropyrimidinyl benzazepine carboxamide, a benzo[b]azepine,benzazepine dicarboxamide derivatives with a tertiary amide, benzazepinedicarboxamide derivatives with a secondary amide, a quinazoline, apyrido[3,2-d]pyrimidine, a diamino-pyrimidine, an amino-quinazoline, aheterocyclic-substituted 2-amino-quinazoline, a diamino-pyrimidine, apiperidino-pyrimidine, an alkylamino-pyrimidine, an 8-substitutedbenzoazepine, an amino-diazepine, an amino-benzo-diazepine, anamido-indole, an amido-benzimidazole, a phenyl sulfonamide, adihydropteridinone, a fused amino-pyrimidine, a quinazoline, apyrido-pyrimidine, an amino-substituted benzazepine, a pyrrolo-pyridine,an imidazo-pyridine derivatives, and an amino-benzazepine, and is otherthan a ssRNA. In some embodiments, a TLR8 agonist is a non-naturallyoccurring compound. Examples of TLR8 agonists include selgantolimod,motolimod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763,VTX-1463, and the TLR8 modulator compounds disclosed in US PatentApplication Publication No. US 2018/0086755 (Gilead,pyrido[3,2-d]pyrimidine derivatives), International Publication No. WO2017216054 (Roche, dihydropyrimidinyl benzazepine carboxamidederivatives), International Publication No. WO 2017/190669 (Shanghai DeNovo Pharmatech, benzo[b]azepine derivatives), International PublicationNo. WO 2016/142250 (Roche, benzazepine dicarboxamide derivatives),International Publication No. WO 2017/202704 (Roche, benzazepinedicarboxamide derivatives with a tertiary amide), InternationalPublication No. WO2 017/202703 (Roche, benzazepine dicarboxamidederivatives with a secondary amide), US Patent Application PublicationNo. US 2017/0071944 (Gilead, quinazoline and pyrido[3,2-d]pyrimdinederivatives), US Patent Application Publication No. US 2014/0045849(Janssen, diamino-pyrimidine derivatives), US Patent ApplicationPublication No. US 2014/0073642 (Janssen, amino-quinazolinederivatives), International Publication No. WO 2014/056953 (Janssen,pyrrolo[3,2-d]pyrimidine derivatives), International Publication No. WO2014/076221 (Janssen, heterocyclic substituted 2-amino-quinazolinederivatives), International Publication No. WO 2014/128189 (Janssen,diamino-pyrimidine derivatives), US Patent Application Publication No.2014/0350031 (Janssen, piperidino-pyrimidine derivatives), InternationalPublication No. WO2014/023813 (Janssen, alkyl-aminopyrimidinederivatives), US Patent Application Publication No. US 2008/0234251(Array Biopharma, 8-substituted benzoazepine derivatives), US PatentApplication Publication No. US 2008/0306050 (Array Biopharma,amino-diazepine derivatives), US Patent Application Publication No. US2010/0029585 (VentiRx Pharma, amino-benzazepine derivatives), US PatentApplication Publication No. US 2011/0092485 (VentiRx Pharma,amino-benzazepine derivatives), US Patent Application Publication No. US2011/0118235 (VentiRx Pharma, amino-benzazepine derivatives), US PatentApplication Publication No. US 2012/0082658 (VentiRx Pharma,amino-benzazepine VTX-378), US Patent Application Publication No. US2012/0219615 (VentiRx Pharma), US Patent Application Publication No. US2014/0066432 (VentiRx Pharma, amino-benzazepine VTX-2337), US PatentApplication Publication No. US 2014/0088085 (VentiRx Pharma,amino-benzazepine and amino-benzo-diazepine derivatives), US PatentApplication Publication No. US 2014/0275167 (Novira Therapeutics,amido-indole and amido-benzimidazole derivatives), and US PatentApplication Publication No. US 2013/0251673 (Novira Therapeutics, phenylsulfonamide derivatives), and these publications are incorporated byreference herein. Further examples of TLR8 modulators include compoundsdisclosed in US Patent Application Publication No. US 2016/0108045(Gilead, dihydropteridinone derivatives), US Patent ApplicationPublication No. US 2018/0065938 (Gilead, fused amino-pyrimidinederivatives), US Patent Application Publication No. US 2018/0263985(Gilead, quinazoline and pyrido-pyrimidine derivatives), InternationalPublication No. WO 2017/046112 (Roche, amino-substituted benzazepinederivatives), International Publication No. WO 2016/096778 (Roche,amino-substituted benzazepine derivatives), US Patent ApplicationPublication No. 2019/0016808 (Birdie Biopharmaceuticals, pyrrolo- orimidazo-pyridine derivatives or amino-benzazepine derivatives), andthese publications are incorporated by reference herein. In someembodiments, the TLR8 agonist comprises the structure:

wherein the structure is optionally substituted at any position otherthan the —NH₂ position. In some embodiments, a TLR8 agonist has an EC50value of 500 nM or less by PBMC assay measuring TNFalpha production. Insome embodiments, a TLR8 agonist has an EC50 value of 100 nM or less byPBMC assay measuring TNFalpha production. In some embodiments, a TLR8agonist has an EC50 value of 50 nM or less by PBMC assay measuringTNFalpha production. In some embodiments, a TLR8 agonist has an EC50value of 10 nM or less by PBMC assay measuring TNFalpha production.

In some embodiments, a TLR8 agonist is a benzazepine selected fromcompounds provided herein (e.g., compounds of Category A and CategoryC).

In some embodiments, a myeloid cell agonist is a ligand of TLR9 selectedfrom the group consisting of: ODN1585, ODN1668, ODN1826, PF-3512676(ODN2006), ODN2007, ODN2216, ODN2336, ODN2395, BB-001, BB-006, CYT-003,IMO-2055, IMO-2125, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod,DIMS-9054, DV-1079, DV-1179, AZD-1419, leftolimod (MGN-1703), litenimod,and CYT-003-QbG10.

In other embodiments, the myeloid agonist selectively agonizes TLR9,TLR3, TLR4, TLR2, TLR5, RIG-I, STING, cGAS, NOD1, NOD2, NOD1/NOD2,NRLP3, ALPK1, MDA5 AIM2, IRE1 and PERK.

In some embodiments, a myeloid cell agonist is a ligand of TLR10.

In some embodiments, a myeloid cell agonist is a ligand of a ligand ofnucleotide-oligomerization domain (NOD)-like selected from the groupconsisting of: NOD1 agonist (C12-iE-DAP, iE-DAP, Tri-DAP), NOD2 agonist(L18-MDP, MDP, M-TriLYS, M-TriLYS-D-ASN, Murabutide, N-Glycolyl-MDP),and NOD1/NOD2 agonists (M-TriDAP, PGN).

In some embodiments, a myeloid cell agonist is a ligand of one or moreRIG-I-Like receptors (RLR) selected from the group consisting of:S′ppp-dsRNA, Poly (dA:dT), Poly(dG:dC), and Poly (I:C).

In some embodiments, a myeloid cell agonist is a ligand of one or moreC-type lectin receptors (CLR) selected from the group consisting of:Cnrdlan AL, HKCA, HKSC, WGP, Zymosan, and Trehalose-6,6-dibehenate.

In some embodiments, a myeloid cell agonist is a ligand of one or moreCytosolic DNA Sensors (CDS) selected from the group consisting of:ADU-S100, c-GMP, c-G-AMP, c-G-GMP, c-A-AMP, c-di-AMP, c-di-IMP,c-di-GMP, c-di-UMP, HSV-60, ISD, pCpG, Poly (dA:dT), Poly(dG:dC), Poly(dA), VACV-70 and α-mangostin and the compounds disclosed inInternational Publication No. WO 2018/156625 (U of Texas), InternationalPublication No. WO 2018/152453 (Eisai), International Publication No. WO2018/138685 (Janssen), International Publication No. WO 2018/100558(Takeda), International Publication No. WO 2018/098203 (Janssen),International Publication No. WO 2018/065360 (Biolog Life Sciences),International Publication No. WO 2018/060323 (Boehringer Ingelheim),International Publication No. WO 2018/045204 (IFM Therapeutics),International Publication No. WO 2018/009466 (Aduro), InternationalPublication No. WO 2017/161349 (Immune Sensor), InternationalPublication No. WO 2017/123669, International Publication No. WO2017/123657, International Publication No. WO 2017/027646 (Merck),International Publication No. WO 2017/027645 (Merck), InternationalPublication No. WO2016/120305 (GSK), International Publication No. WO2016/096174 (InvivoGen), and US Patent Application Publication No. US2014/0341976 (Aduro).

In some embodiments, the myeloid cell agonist is a ligand of aninflammasome inducer selected from the group consisting of: (a) NLRP3inflammasome protein complex, preferably alum Crystals, ATP, CPPDCrystals, Hennozoin, MSU Crystals, Nano-Si 02, Nigericin, and (b) AIM2inflammasome protein complex, such as Poly (dA:dT).

In certain aspects, a TLR8 agonist is selected from Category A orCategory C, or a TLR7 agonist is selected from Category B, as furtherdescribed herein. Variables and Formula of the Compounds of Category A(TLR8 agonists) are described in the section entitled Compounds ofCategory A; variables and Formula of the Compounds of Category B (TLR7agonists) are described in section entitled Compounds of Category B; andvariables and Formula of the Compounds of Category C (TLR8 agonists) aredescribed in section entitled Compounds of Category C. Formulas andvariables of the Compounds of Category A, the Compounds of Category Band the Compounds of Category C may overlap in nomenclature, e.g.,Formula IA for Compounds of each of Category A, Category B and CategoryC; however, variables and Formula descriptions are not intended to beinterchangeable between the categories.

Compounds of Category A. TLR8 Agonists

In some aspects, the myeloid cell agonist is a benzazepine-4-carboxamidecompound. In certain embodiments, the benzazepine-4-carboxamide compoundhas the structure of Formula X-1:

wherein:

R¹ is C₃₋₇ alkyl;

R² is C₃₋₇ alkyl or C₃₋₇ cycloalkyl-C₁₋₇ alkyl;

R³ is hydrogen;

R⁴ is selected from the group consisting of

C₁₋₇ alkyl, said C₁₋₇ alkyl being unsubstituted or substituted by one ortwo groups selected from the group consisting of phenyl and heteroaryl,said heteroaryl being an aromatic 5- or 6-membered ring which comprisesone, two, or three atoms selected from nitrogen, oxygen, and/or sulfur;

C₃₋₇ cycloalkyl, said C₃₋₇ cycloalkyl being unsubstituted or substitutedby phenyl or phenylamino-C₁₋₄ alkyl, and

heterocyclyl, said heterocyclyl being a saturated 3- to 7-membered ringcontaining one heteroatom selected from N and O and being unsubstitutedor substituted by phenyl.

Structures of Formula X-1 are described, for example, in InternationalPublication No. WO 2017/202703.

In some aspects, the myeloid cell agonist is a benzazepine-dicarboxamidecompound. In certain embodiments, the benzazepine-dicarboxamide compoundhas the structure of Formula X-2:

wherein:

R¹ is C₃₋₇ alkyl;

R² is C₃₋₇ alkyl or C₃₋₇ cycloalkyl-C₁₋₇ alkyl;

R³ is a heterocycle selected from

wherein

X₁ is (CH₂)_(m) wherein m is 1 or 2;

X₂ is (CH₂)_(n) wherein n is 1 or 2;

X₃ is (CH₂)_(o) wherein o is 1 or 2;

X₄ is (CH₂)_(P) wherein p is 1 or 2; and

Z₁ is phenyl, wherein phenyl is unsubstituted or substituted by one ortwo groups selected from the group consisting of C₁₋₇ alkyl, halogen,halogen-C₁₋₇ alkyl, C₁₋₇ alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl,C₁₋₇ alkyl-amino-C₁₋₇ alkyl, and di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl; or

wherein

X₅ is (CH₂)_(q) wherein q is 1 or 2;

X₆ is (CH₂)_(r) wherein r is 1 or 2;

Y₁ is a carbon or nitrogen atom;

Z₂ is hydrogen; and

Z₃ is selected from the group consisting of hydrogen, C₁₋₇ alkoxy, C₂₋₇alkenyloxy, phenyl, phenyl-C₁₋₇ alkyl, phenyl-C₁₋₇ alkyloxy, phenyl-C₁₋₇alkylamino, phenylamino-C₁₋₇ alkyl, phenylamino, wherein phenyl isunsubstituted or substituted by one or two groups selected from thegroup consisting of C₁₋₇ alkyl, halogen, halogen-C₁₋₇ alkyl, C₁₋₇alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl, C₁₋₇ alkyl-amino-C₁₋₇alkyl, and di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl; or

wherein

X₇ is (CH₂)_(s) wherein s is 1 or 2; and

Z₄ is phenyl, wherein phenyl is unsubstituted or substituted by one ortwo groups selected from the group consisting of C₁₋₇ alkyl, halogen,halogen-C₁₋₇ alkyl, C₁₋₇ alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl,C₁₋₇alkyl-amino-C₁₋₇ alkyl, and di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl; or

wherein

X₈ is (CH₂)_(t) wherein t is 1 or 2; and

Z₅ is phenyl, wherein phenyl is unsubstituted or substituted by one ortwo groups selected from the group consisting of C₁₋₇ alkyl, halogen,halogen-C₁₋₇ alkyl, C₁₋₇ alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl,C₁₋₇ alkyl-amino-C₁₋₇ alkyl, and di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl.

Compounds of Formula X-2 are described, for example, in InternationalPublication No. WO 2017/202704.

In some aspects, the myeloid cell agonist is a benzazepine sulfonamidecompound. In certain embodiments, the benzazepine sulfonamide compoundhas the structure of Formula X-3:

wherein

R¹ and R² are the same or different and are selected from the groupconsisting of C₁₋₇ alkyl, hydroxy-C₂₋₇ alkyl, amino-C₂₋₇ alkyl, C₂₋₇alkenyl, and C₃₋₇ alkynyl;

R³ is hydrogen or C₁₋₇ alkyl;

R⁶ is hydrogen or C₁₋₇ alkyl;

one of R⁴ and R⁵ is selected from the group consisting of hydrogen, C₁₋₇alkyl, halogen-C₁₋₇ alkyl, and C₁₋₇ alkoxy,

and the other one of R⁴ and R⁵ is

wherein R⁷ and R⁸ are the same or different and are selected from thegroup consisting of hydrogen, C₁₋₇ alkyl, halogen-C₁₋₇ alkyl,hydroxy-C₁₋₇ alkyl, hydroxy-C₁₋₇ alkoxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl,C₁₋₇ alkyl-amino-C₁₋₇ alkyl, amino-C₁₋₇ alkoxy-C₁₋₇ alkyl, C₁₋₇alkyl-amino-C₁₋₇ alkoxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl-carbonyl, and C₁₋₇alkyl-xamino-C₁₋₇ alkyl-carbonyl; or

R⁷ and R⁸ together with the nitrogen atom they are attached to form a 4-to 6-membered heterocycle which is unsubstituted or substituted with agroup selected from the group consisting of amino, C₁₋₇ alkyl-amino,hydroxy, and hydroxy-C₁₋₇ alkyl, and which may contain an additionalN—R¹⁰ group, wherein R¹⁰ is selected from the group consisting ofhydrogen, amino-C₁₋₇ alkyl, and C₁₋₇ alkyl-amino-C₁₋₇ alkyl; and

Y is N or CR⁹;

wherein R⁹ is selected from the group consisting of hydrogen, C₁₋₇alkyl, and halogen-C₁₋₇ alkyl.

Compounds of Formula X-3 are described, for example, in InternationalPublication No. WO 2016/096778.

In some aspects, the myeloid cell agonist is a dihydropyrimidinylbenzazepine carboxamide compound. In some aspects, thedihydropyrimidinyl benzazepine carboxamide compound has the structure ofFormula X-4:

wherein

R¹ is C₃₋₇ alkyl;

R² is C₃₋₇ alkyl or C₃₋₇ cycloalkyl-C₁₋₇ alkyl;

R³ is hydrogen or C₁₋₇ alkyl;

R⁴ is hydrogen or C₁₋₇ alkyl;

R⁵ is selected from the group consisting of hydrogen, halogen, C₁₋₇alkyl, and C₁₋₇ alkoxy;

R⁶ is selected from the group consisting of hydrogen, halogen, C₁₋₇alkyl, and C₁₋₇ alkoxy; and

X is N or CR⁷, wherein R⁷ is selected from the group consisting ofhydrogen, halogen, C₁₋₇ alkyl, and C₁₋₇ alkoxy.

Compounds of Formula X-4 are described, for example, in InternationalPublication No. WO2017/216054.

In some aspects, the myeloid cell agonist is a sulfinylphenyl orsulfonimidoylphenyl benzazepine compound. In some aspects, thesulfinylphenyl or sulfonimidoylphenyl benzazepine compound has thestructure of Formula X-5:

wherein

X is CR⁷ or N;

R¹ is C₃₋₇ alkyl or C₃₋₇ cycloalkyl;

R² is selected from the group consisting of C₃₋₇ alkyl, hydroxy-C₁₋₇alkyl, C₃₋₇-alkynyl, amino-C₁₋₇ alkoxy-C₁₋₇ alkoxy-C₁₋₇ alkyl,halogen-C₁₋₇ alkyl, and C₃₋₇ cycloalkyl-C₁₋₇ alkyl;

one of R³ and R⁴ is

and the other one of R³ and R⁴ is selected from the group consisting ofhydrogen, C₁₋₇ alkyl, and halogen;

R⁵, R⁶, and R⁷ are independently from each other selected from hydrogen,C₁₋₇ alkyl, and halogen;

R⁸ is C₁₋₇ alkyl; and

R⁹ is absent or is ═N—R¹⁰, wherein R¹⁰ is selected from the groupconsisting of hydrogen, C₁₋₇ alkyl, halogen-C₁₋₇ alkyl, hydroxy-C₁₋₇alkyl, and hydroxy-C₁₋₇ alkoxy-C₁₋₇ alkyl.

Compounds of Formula X-5 are described, for example, in InternationalPublication No. WO 2017/046112.

In some aspects, the myeloid cell agonist is a TLR modulator compoundthat has the structure of Formula X-6:

wherein

(1) is a double bond or a single bond;

(2) is a single bond or is double bond and R₁ is absent;

-   -   R² and R₃ are independently selected from H and lower alkyl, or        R₂ and R₃ are connected to form a saturated carbocycle having        from 3 to 7 ring members; one of R₇ and R₈ is —NR_(f)R_(g),

-   -    and    -   the other is hydrogen;    -   where R_(f) and R_(g) are lower alkyl or R_(f) and R_(g)        together with the nitrogen to which they are attached form a        saturated heterocyclic ring having 4 to 6 ring members;    -   R₄ is —NR_(c)R_(a) or —OR₁₀;        -   R_(c) and Rd are lower alkyl, where the alkyl is optionally            substituted with one or more —OH;        -   R₁₀ is alkyl, where the alkyl is optionally substituted with            one or more —OH;    -   Z is C and        (1) is a double bond, or Z is N and        (1) is a single bond;    -   R_(a) and R_(b) are independently selected from H, alkyl,        alkenyl, alkynyl, and R^(e),    -   wherein the alkyl is optionally substituted with one or more        —OR¹⁰, or R^(e);    -   R^(e) is selected from —NH₂, —NH(alkyl), and —N(alkyl)₂;    -   R¹ is absent when        (2) is a double bond, or when        (2) is a single bond, R¹ and        one of R^(a) or R^(b) are taken together with the atoms to which        they are attached to form a saturated, partially unsaturated, or        unsaturated heterocycle having 5-7 ring members, and the other        of R^(a) or R^(b) is hydrogen or is absent as necessary to        accommodate ring unsaturation.

In some aspects, the myeloid cell agonist is a TLR modulator compoundthat has the structure of Formula X-7:

wherein

Y is CF₂CF₃, CF₂CF₇R⁶, or an aryl or heteroaryl ring, wherein said aryland heteroaryl rings are substituted with one or more groupsindependently selected from alkenyl, alkynyl, Br, CN, OH, NR⁶R⁷,C(═O)R⁸, NR⁶SO₂R⁷, (C₁-C₆ alkyl)amino, R⁶OC(═O)CH═CH₂—, SR⁶ and SO₂R⁶,and wherein the aryl and heteroaryl rings are optionally furthersubstituted with one or more groups independently selected from F, Cl,CF₃, CF₃O—, HCF₂O—, alkyl, heteroalkyl and ArO—;

R¹, R³ and R⁴ are independently selected from H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R³ and R⁴ together with the atom to which they are attached form asaturated or partially unsaturated carbocyclic ring, wherein thecarbocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

R² and R⁸ are independently selected from H, OR⁶, NR⁶R⁷, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

R^(5a), R^(5b), and R^(5c) are independently H, F, Cl, Br, I, OMe, CH₃,CH₂F, CHF₂ or CF3; and

R⁶ and R⁷ are independently selected from H, alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl andheteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R⁶ and R⁷ together with the atom to which they are attached form asaturated or partially unsaturated heterocyclic ring, wherein saidheterocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶.

In some aspects, the myeloid cell agonist is a TLR modulator compoundthat has the structure of Formula X-8:

wherein

-   -   W is —C(O)—;    -   Z is H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, OR⁶ or NR⁶R⁷, wherein the        alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, aryl and heteroaryl are optionally substituted        with one or more groups independently selected from alkyl,        alkenyl, alkynyl. F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,        C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,        R⁶OCC═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;    -   R¹, R², R³ and R⁴ are independently selected from H, alkyl,        alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl,        heterocycloalkyl, aryl and heteroaryl, wherein said alkyl,        alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl,        heterocycloalkyl, aryl, and heteroaryl are optionally        substituted with one or more groups independently selected from        alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,        C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,        R⁶OC(C═O)CH═CH₂—, NR⁶SO₂R⁷, SR₆ and SO₂R⁶,    -   or R¹ and R² together with the atom to which they are attached        form a saturated or partially unsaturated carbocyclic ring,        wherein said carbocyclic ring is optionally substituted with one        or more groups independently selected from alkyl, alkenyl,        alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶,        OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,        R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,    -   or R³ and R⁴ together are oxo;    -   R⁵ is H, F, Cl, Br, I, OMe, CH₃, CH₂F, CHF₂, CF₃ or CF₂CF₃;    -   R⁶ and R⁷ are independently selected from H, alkyl, alkenyl,        alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl,        heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl,        alkenyl, alkynyl, heteroalkyl, cycloalkyl cycloalkenyl,        heterocycloalkyl, aryl, and heteroaryl are optionally        substituted with one or more groups independently selected from        alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,        C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,        R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;    -   or R⁶ and R⁷ together with the atom to which they are attached        form a saturated or partially unsaturated heterocyclic ring,        wherein said heterocyclic ring is optionally substituted with        one or more groups independently selected from alkyl, alkenyl,        alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶,        OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,        R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶; and    -   n is 0, 1, 2, 3 or 4.

Compounds of Formula X-6, X-7, and X-8 are described, for example, inU.S. Publication Nos. US 2019/0016808 and US 2014/0088085.

In some aspects, the myeloid cell agonist is a TLR modulator compoundthat has the structure of Formula X-9:

whereinR¹ is C₃₋₇ alkyl or C₃₋₇ cycloalkyl;R² is selected from the group consisting of C₁₋₇ alkyl, hydroxy-C₁₋₇alkyl, C₂₋₇ alkenyl, C₃₋₇ alkynyl, amino-C₁₋₇ alkoxy-C₁₋₇alkyl,amino-C₁₋₇ alkoxy-C₁₋₇alkoxy-C₁₋₇ alkyl, halogen-C₁₋₇ alkyl, C₃₋₇cycloalkyl-C₁₋₇ alkyl, and phenyl-C₁₋₇ alkyl, wherein phenyl isunsubstituted or substituted by amino-C₁₋₇ alkyl;R³ is hydrogen;R⁴ is selected from the group consisting of

phenyl, said phenyl being unsubstituted or substituted by one or twogroups selected from the group consisting of C₁₋₇ alkyl, halogen,halogen-C₁₋₇ alkyl, C₁₋₇ alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl,C₁₋₇ alkyl-amino-C₁₋₇ alkyl, di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl, amino-C₂₋₇alkenyl, C₁₋₇ alkyl-amino-C₂₋₇ alkenyl, di-C₁₋₇ alkyl-amino-C₂₋₇alkenyl, amino-C₂₋₇ alkynyl, C₁₋₇ alkyl-amino-C₂₋₇ alkynyl, di-C₁₋₇alkyl-amino-C₂₋₇ alkynyl, benzyloxycarbonylamino-C₁₋₇ alkyl, amino-C₁₋₇alkoxy, amino-C₁₋₇ alkoxy-C₁₋₇ alkoxy, amino-C₁₋₇ alkoxy-C₁₋₇ alkyl,amino-C₁₋₇ alkoxy-C₁₋₇ alkoxy-C₁₋₇ alkyl, C₁₋₇ alkylsulfonyl,heterocyclylcarbonyl, and phenyl-C₁₋₇ alkyl, wherein phenyl isunsubstituted or substituted by C₁₋₇ alkoxy or amino-C₁₋₇ alkyl; or

heteroaryl, said heteroaryl being a 5- or 6-membered aromatic ringcontaining one, two, or three heteroatoms selected from N, O, or S, andbeing unsubstituted or substituted by one or two groups selected fromthe group consisting of C₁₋₇ alkyl, halogen, halogen-C₁₋₇ alkyl, C₁₋₇alkoxy, hydroxy-C₁₋₇ alkyl, amino-C₁₋₇ alkyl, C₁₋₇ alkyl-amino-C₁₋₇alkyl, di-C₁₋₇ alkyl-amino-C₁₋₇ alkyl, amino-C₂₋₇ alkenyl, C₁₋₇alkyl-amino-C₂₋₇ alkenyl, di-C₁₋₇ alkyl-amino-C₂₋₇ alkenyl, amino-C₂₋₇alkynyl, C₁₋₇ alkyl-amino-C₂₋₇ alkynyl, di-C₁₋₇ alkyl-amino-C₂₋₇alkynyl, benzyloxycarbonylamino-C₁₋₇ alkyl, amino-C₁₋₇ alkoxy,amino-C₁₋₇ alkoxy-C₁₋₇ alkoxy, amino-C₁₋₇ alkoxy-C₁₋₇ alkyl, amino-C₁₋₇alkoxy-C₁₋₇ alkoxy-C₁₋₇ alkyl, C₁₋₇ alkylsulfonyl, heterocyclylcarbonyl,and phenyl-C₁₋₇ alkyl, wherein phenyl is unsubstituted or substituted byC₁₋₇ alkoxy or amino-C₁₋₇ alkyl.

Compounds of Formula X-9 are described, for example, in InternationalPublication No. WO 2016/142250.

In some aspects, the present disclosure provides a TLR8 agonistrepresented by the structure of Formula (IIA):

or a pharmaceutically acceptable salt thereof, wherein:

represents an optional double bond;

L¹⁰ is —X¹⁰—;

L² is selected from —X²—, —X²—C₁₋₆alkylene-X²—, —X²—C₂₋₆ alkenylene-X²—,and —X²—C₂₋₆ alkynylene-X²—, each of which is optionally substituted onalkylene, alkenylene or alkynylene with one or more R¹²;

X¹⁰ is selected from —C(O)—, and —C(O)N(R¹⁰)—*, wherein * representswhere X¹⁰ is bound to R⁵;

X² at each occurrence is independently selected from a bond, —O—, —S—,—N(R¹⁰)—, —C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R¹⁰)—,—C(O)N(R¹⁰)C(O)—, —C(O)N(R¹⁰)C(O)N(R¹⁰), —N(R¹⁰)C(O)—,—N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)O—, —OC(O)N(R¹⁰)—, —C(NR¹⁰)—,—N(R¹⁰)C(NR¹⁰)—, —C(NR¹⁰)N(R¹⁰)—, —N(R¹⁰)C(NR¹⁰)N(R¹⁰)—, —S(O)₂—,—OS(O)—, —S(O)O—, —S(O), —OS(O)₂—, —S(O)₂O, —N(R¹⁰)S(O)₂—,—S(O)₂N(R¹⁰)—, —N(R¹⁰)S(O)—, —S(O)N(R¹⁰)—, —N(R¹⁰)S(O)₂N(R¹⁰)—, and—N(R¹⁰)S(O)N(R¹⁰)—;

R¹ and R² are independently selected from hydrogen; and C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰,—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN;

R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle in R⁴ is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl;

R⁵ is selected from unsaturated C₄₋₈ carbocycle; bicyclic carbocycle;and fused 5-5, fused 5-6, and fused 6-6 bicyclic heterocycle, wherein R⁵is optionally substituted and wherein substituents are independentlyselected at each occurrence from: halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle in R⁵ is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆alkynyl;

R¹⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋to alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, —C₁₋₁₀ haloalkyl, —O—C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂ carbocycle, 3- to 12-membered heterocycle,and haloalkyl; and

R¹² is independently selected at each occurrence from halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀carbocycle and 3- to 10-membered heterocycle; and C₃₋₁₀ carbocycle and3- to 10-membered heterocycle, wherein each C₃₋₁₀ carbocycle and 3- to10-membered heterocycle in R¹² is optionally substituted with one ormore substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl;

wherein any substitutable carbon on the benzazepine core is optionallysubstituted by a substituent independently selected from R¹² or twosubstituents on a single carbon atom combine to form a 3- to 7-memberedcarbocycle.

In some embodiments, the compound of Formula (IIA) is represented byFormula (IIB):

or a pharmaceutically acceptable salt thereof, wherein:

R²⁰, R²¹, R²², and R²³ are independently selected from hydrogen,halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl; and

R²⁴ and R²⁵ are independently selected from hydrogen, halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; or R²⁴ and R²⁵ taken together form an optionally substitutedsaturated C₃₋₇ carbocycle.

In some embodiments, R²⁰, R²¹, R²², and R²³ are independently selectedfrom hydrogen, halogen, —OH, —OR¹⁰, —NO₂, —CN, and C₁₋₁₀ alkyl. R²⁰,R²¹, R²², and R²³ may be each hydrogen. In certain embodiments, R²¹ ishalogen. In certain embodiments, R²¹ is hydrogen. In certainembodiments, R²¹ is —OR¹⁰. For example, R²¹ may be —OCH₃.

In some embodiments, R²⁴ and R²⁵ are independently selected fromhydrogen, halogen, —OH, —NO₂, —CN, and C₁₋₁₀ alkyl, or R²⁴ and R²⁵ takentogether form an optionally substituted saturated C₃₋₇ carbocycle. Incertain embodiments, R²⁴ and R²⁵ are each hydrogen. In otherembodiments, R²⁴ and R²⁵ taken together form an optionally substitutedsaturated C₃₋₅ carbocycle, wherein substituents are selected fromhalogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and—CN; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which isindependently optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle.

In some embodiments, R¹ is hydrogen. In some embodiments, R² ishydrogen. In some embodiments, R² is-C(O)—.

In some embodiments, L¹⁰ is selected from —C(O)N(R¹⁰)—*. In certainembodiments, R¹⁰ of —C(O)N(R¹⁰)—* is selected from hydrogen and C₁₋₆alkyl. For example, L¹⁰ may be —C(O)NH—*.

In some embodiments, R⁵ is an optionally substituted bicycliccarbocycle. In certain embodiments, R⁵ is an optionally substituted 8-to 12-membered bicyclic carbocycle. R⁵ may be an optionally substituted8- to 12-membered bicyclic carbocycle substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, —CN, C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl. In certain embodiments, R⁵ is anoptionally substituted 8- to 12-membered bicyclic carbocycle substitutedwith one or more substituents independently selected from —OR¹⁰,—N(R¹⁰)₂, and ═O. In some embodiments, R⁵ is an optionally substitutedindane, and optionally substituted tetrahydronaphthalene. R⁵ may beselected from:

any one of which is optionally substituted. For example, the R⁵ isselected from:

In some embodiments, R⁵ is an optionally substituted unsaturated C₄₋₈carbocycle. In certain embodiments, R⁵ is an optionally substitutedunsaturated C₄₋₆ carbocycle. In certain embodiments, R⁵ is an optionallysubstituted unsaturated C₄₋₆ carbocycle with one or more substituentsindependently selected from optionally substituted C₃₋₁₂ carbocycle, andoptionally substituted 3- to 12-membered heterocycle. R⁵ may be anoptionally substituted unsaturated C₄₋₆ carbocycle with one or moresubstituents independently selected from optionally substituted phenyl,optionally substituted 3- to 12-heterocycle, optionally substitutedC₁₋₁₀ alkyl, optionally substituted C₂₋₁₀ alkenyl, and halogen.

In some embodiments, R⁵ is selected from an optionally substituted fused5-5, fused 5-6, and fused 6-6 bicyclic heterocycle. In certainembodiments, R⁵ is an optionally substituted fused 5-5, fused 5-6, andfused 6-6 bicyclic heterocycle with one or more substituentsindependently selected from —C(O)OR¹⁰, —N(R¹⁰)₂, —OR¹⁰, and optionallysubstituted C₁₋₁₀ alkyl. In certain embodiments, R⁵ is an optionallysubstituted fused 5-5, fused 5-6, and fused 6-6 bicyclic heterocyclesubstituted with —C(O)OR¹⁰. In certain embodiments, R⁵ is an optionallysubstituted fused 6-6 bicyclic heterocycle. For example, the fused 6-6bicyclic heterocycle may be an optionally substitutedpyridine-piperidine. In some embodiments, L¹⁰ is bound to a carbon atomof the pyridine of the fused pyridine-piperidine. In certainembodiments, R⁵ is selected from tetrahydroquinoline,tetrahydroisoquinoline, tetrahydronaphthyridine, cyclopentapyridine, anddihydrobenzoxaborole, any one of which is optionally substituted. R⁵ maybe an optionally substituted tetrahydronaphthyridine. In someembodiments, R⁵ is selected from:

In some embodiments, when R⁵ is substituted, substituents on R⁵ areindependently selected at each occurrence from: halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OR¹⁰,—SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆alkynyl. In certain embodiments, the substituentson R⁵ are independently selected at each occurrence from: halogen,—OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and—CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O,═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle.In certain embodiments, the substituents on R⁵ are independentlyselected at each occurrence from: halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, and—CN; and C₁₋₁₀ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —NO₂, ═O, and —CN. In some embodiments,R⁵ is not substituted.

In some embodiments, L² is selected from —C(O)—, and —C(O)NR¹⁰—. In someembodiments, L² is —C(O)—. In some embodiments, L² is —C(O)NR¹⁰—. R¹⁰ of—C(O)NR¹⁰— may be selected from hydrogen and C₁₋₆ alkyl. For example, L²may be —C(O)NH—.

In some embodiments, R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R₁₀)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle and 3- to12-membered, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl. In some embodiments, R⁴ is selected from:—OR¹⁰, and —N(R¹⁰)₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰,—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl. In certain embodiments, R⁴ is—N(R¹⁰)₂. R¹⁰ of —N(R¹⁰)₂ may be independently selected at eachoccurrence from optionally substituted C₁₋₆ alkyl. In certainembodiments, R¹⁰ of —N(R¹⁰)₂ is independently selected at eachoccurrence from methyl, ethyl, propyl, and butyl, any one of which isoptionally substituted. For example, R⁴ may be

In certain embodiments, -L²-R⁴ is

In some embodiments, R¹² is independently selected at each occurrencefrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)_(2I)—NO₂, ═O, ═S,═N(R¹⁰), —CN, C₃₋₁₀ carbocycle and 3- to 10-membered heterocycle; andC₃₋₁₀ carbocycle and 3- to 10-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R₁₀, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl. In certain embodiments, R¹² is independently selected ateach occurrence from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; and C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂,—NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀ carbocycle and 3- to 10-memberedheterocycle.

In some embodiments, the compound of Formula (IIB) is a compound ofFormula (IIC):

or a pharmaceutically acceptable salt thereof,

wherein:

R¹ and R² are hydrogen;

L² is —C(O)—;

R⁴ is —N(R¹⁰)₂;

R¹⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, —O, —S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, —C₁₋₁₀ haloalkyl, —O—C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂ carbocycle, 3- to 12-membered heterocycle,and haloalkyl;

L¹⁰ is —C(O)N(R¹⁰)—*, wherein * represents where L¹⁰ is bound to R⁵; and

R⁵ is a fused 5-5, fused 5-6, or fused 6-6 bicyclic heterocycle, whereinR⁵ is optionally substituted and wherein substituents are independentlyselected at each occurrence from:

halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and—CN;

C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle; and

C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O,═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl.

In certain embodiments, R¹⁰ of —N(R¹⁰)₂ is independently selected ateach occurrence from methyl, ethyl, propyl, and butyl, any one of whichis optionally substituted; and/or R¹⁰ of —C(O)N(R¹⁰)—* is hydrogen.

In certain embodiments, R⁴ is

and/or R¹⁰ of —C(O)N(R¹⁰)—* is hydrogen.

In some embodiments, the compound is selected from:

and a salt of any one thereof.

In some aspects, the present disclosure provides a compound representedby the structure of Formula (IIIA):

or a pharmaceutically acceptable salt thereof, wherein:

represents an optional double bond;

L¹¹ is —X¹¹—;

L² is selected from —X²—, —X²—C₁₋₆alkylene-X²—, —X²—C₂₋₆ alkenylene-X²—,and —X²—C₂₋₆ alkynylene-X²—, each of which is optionally substituted onalkylene, alkenylene or alkynylene with one or more R¹²;

X¹¹ is selected from —C(O)— and —C(O)N(R¹⁰)—*, wherein * representswhere X¹¹ is bound to R⁶;

X² at each occurrence is independently selected from a bond, —O—, —S—,—N(R¹⁰)—, —C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R¹⁰)—,—C(O)N(R¹⁰)C(O)—, —C(O)N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)—,—N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)O—, —OC(O)N(R¹⁰)—, —C(NR¹⁰)—,—N(R¹⁰)C(NR¹⁰)—, —C(NR¹⁰)N(R¹⁰)—, —N(R¹⁰)C(NR¹⁰)N(R¹⁰)—, —S(O)₂—,—OS(O)—, —S(O)O—, —S(O)—, —OS(O)₂—, —S(O)₂O—, —N(R¹⁰)S(O)₂—,—S(O)₂N(R¹⁰)—, —N(R¹⁰)S(O)—, —S(O)N(R¹⁰)—, —N(R¹⁰)S(O)₂N(R¹⁰)—, and—N(R¹⁰)S(O)N(R¹⁰)—;

R¹ and R² are independently selected from hydrogen; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl, each of which is optionally substituted withone or more substituents independently selected from halogen, —OR¹⁰,—SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —S(O)R, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN;

R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle in R⁴ is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl,C₂₋₆alkenyl, and C₂₋₆ alkynyl;

R⁶ is selected from phenyl and 5- or 6-membered heteroaryl, any one ofwhich is substituted with one or more substituents selected from R⁷ andR⁶ is further optionally substituted by one or more additionalsubstituents independently selected from R¹²;

R⁷ is selected from —C(O)NHNH₂, —C(O)NH—C₁₋₃ alkylene-NH(R¹⁰), —C(O)CH₃,—C₁₋₃ alkylene-NHC(O)OR¹¹, —C₁₋₃ alkylene-NHC(O)R¹⁰, —C₁₋₃alkylene-NHC(O)NHR¹⁰, —C₁₋₃ alkylene-NHC(O)—C₁₋₃ alkylene-R¹⁰, and a 3-to 12-membered heterocycle optionally substituted with one or moresubstituents independently selected from R¹²;

R¹⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, —C₁₋₁₀ alkyl, —C₁₋₁₀haloalkyl, —O—C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle;

R¹¹ is selected from C₃₋₁₂ carbocycle and 3- to 12-membered heterocycle,each of which is optionally substituted with one or more substituentsindependently selected from R¹²; and

R¹² is independently selected at each occurrence from halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀carbocycle and 3- to 10-membered heterocycle; and C₃₋₁₀ carbocycle and3- to 10-membered heterocycle, wherein each C₃₋₁₀ carbocycle and 3- to10-membered heterocycle in R¹² is optionally substituted with one ormore substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl; and

wherein any substitutable carbon on the benzazepine core is optionallysubstituted by a substituent independently selected from R¹² or twosubstituents on a single carbon atom combine to form a 3- to 7-memberedcarbocycle.

In some embodiments, the compound of Formula (IIIA) is represented byFormula (IIIB):

or a pharmaceutically acceptable salt thereof, wherein:

R²⁰, R²¹, R²², and R²³ are independently selected from hydrogen,halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl; and

R²⁴ and R²⁵ are independently selected from hydrogen, halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; or R²⁴ and R²⁵ taken together form an optionally substitutedsaturated C₃₋₇ carbocycle.

In some embodiments, R²⁰, R²¹, R²², and R²³ are independently selectedfrom hydrogen, halogen, —OH, —NO₂, —CN, and C₁₋₁₀ alkyl. In certainembodiments, R²⁰, R²¹, R²², and R²³ are each hydrogen. In someembodiments, R²⁴ and R²⁵ are independently selected from hydrogen,halogen, —OH, —NO₂, —CN, and C₁₋₁₀ alkyl, or R and R taken together forman optionally substituted saturated C₃₋₇ carbocycle. In certainembodiments, R²⁴ and R²⁵ are each hydrogen. In certain embodiments, R²⁴and R²⁵ taken together form an optionally substituted saturated C₃₋₅carbocycle.

In some embodiments, R¹ is hydrogen. In some embodiments, R² ishydrogen.

In some embodiments, L¹¹ is selected from —C(O)N(R¹⁰)—*. In someembodiments, R¹⁰ of —C(O)N(R¹⁰)—* is selected from hydrogen and C₁₋₆alkyl. For example, L¹¹ may be —C(O)NH—*.

In some embodiments, R⁶ is phenyl substituted with R⁷ and R⁶ is furtheroptionally substituted with one or more additional substituentsindependently selected from R¹². In some embodiments, R⁶ is selectedfrom phenyl substituted with one or more substituents independentlyselected from —C(O)NHNH₂, —C(O)NH—C₁₋₃ alkylene-NH(R¹⁰), —C₁₋₃alkylene-NHC(O)R¹⁰, and —C(O)CH₃; and 3- to 12-membered heterocycle,which is optionally substituted with one or more substituents selectedfrom —OH, —N(R¹⁰)₂, —NHC(O)(R¹⁰), —NHC(O)O(R¹⁰), —NHC(O)N(R¹⁰)₂,—C(O)R¹⁰, —C(O)N(R¹⁰)₂, —C(O)₂R¹⁰, and —C₁₋₃ alkylene-(R¹⁰) and R⁶ isfurther optionally substituted with one or more additional substituentsindependently selected from R¹². For example, R⁶ may be selected from:

In some embodiments, R⁶ is selected from a 5- and 6-membered heteroarylsubstituted with one or more substituents independently selected fromR⁷, and R⁶ is further optionally substituted with one or more additionalsubstituents selected from R¹². In certain embodiments, R⁶ is selectedfrom 5- and 6-membered heteroaryl substituted with one or moresubstituents independently selected from —C(O)CH₃, —C₁₋₃alkylene-NHC(O)OR¹⁰, —C₁₋₃ alkylene-NHC(O)R¹⁰, —C₁₋₃alkylene-NHC(O)NHR¹⁰, and —C₁₋₃ alkylene-NHC(O)—C₁₋₃ alkylene-(R¹⁰); and3- to 12-membered heterocycle, which is optionally substituted with oneor more substituents selected from —OH, —N(R¹⁰)₂, —NHC(O)(R¹⁰),—NHC(O)O(R¹⁰), —NHC(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —C(O)₂R¹⁰, and—C₁₋₃ alkylene-(R¹⁰), and R⁶ is optionally further substituted with oneor more additional substituents independently selected from R¹². R⁶ maybe selected from substituted pyridine, pyrazine, pyrimidine, pyridazine,furan, pyran, oxazole, thiazole, imidazole, pyrazole, oxadiazole,oxathiazole, and triazole, and R⁶ is optionally further substituted withone or more additional substituents independently selected from R¹². Insome embodiments, R⁶ is substituted pyridine and R⁶ is optionallyfurther substituted with one or more additional substituentsindependently selected from R¹². R⁶ may be represented as follows:

In some embodiments, R⁶ is substituted pyridine, and wherein R⁷ is —C₁₋₃alkylene-NHC(O)—C₁₋₃ alkylene-R¹⁰. In certain embodiments, R⁷ is —C₁alkylene-NHC(O)—C₁ alkylene-R¹⁰. In certain embodiments, R⁷ is —C₁alkylene-NHC(O)—C₁ alkylene-NH₂. In some embodiments, R⁶ is selectedfrom:

In certain embodiments, R⁶ is

In some embodiments, L² is selected from —C(O)—, and —C(O)NR¹⁰—. In someembodiments, L² is selected from —C(O)NR¹⁰—. R¹⁰ of —C(O)NR¹⁰— may beselected from hydrogen and C₁₋₆ alkyl. For example, L² may be —C(O)NH—.In some embodiments, L² is —C(O)—.

In some embodiments, R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle and 3- to12-membered, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl. In some embodiments, R⁴ is selected from:—OR¹⁰ and —N(R¹⁰)₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle and 3- to 12-membered heterocycle, each of which isindependently optionally substituted at each occurrence with one or moresubstituents selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰,—S(O)₂R¹⁰—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN,C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl. In certain embodiments,R⁴ is —N(R¹⁰)₂. R¹⁰ of —N(R¹⁰)₂ may be independently selected at eachoccurrence from optionally substituted C₁₋₆ alkyl. In some embodiments,R¹⁰ of —N(R¹⁰)₂ is independently selected at each occurrence frommethyl, ethyl, propyl, and butyl, any of which are optionallysubstituted. For example, R⁴ may be

In some embodiments, -L²-R⁴ is

In some embodiments, R¹² is independently selected at each occurrencefrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is independently optionallysubstituted at each occurrence with one or more substituents selectedfrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀ carbocycle and 3- to10-membered heterocycle; and C₃₋₁₀ carbocycle and 3- to 10-memberedheterocycle, each of which is independently optionally substituted ateach occurrence with one or more substituents selected from halogen,—OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂,—NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl.In certain embodiments, R¹² is independently selected at each occurrencefrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is independently optionallysubstituted at each occurrence with one or more substituents selectedfrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀ carbocycle and 3- to10-membered heterocycle.

In some embodiments, the compound is selected from:

and a salt of any one thereof.

In some aspects, the present disclosure provides a compound representedby the structure of Formula (IA):

or a pharmaceutically acceptable salt thereof, wherein:

represents an optional double bond;

L¹ is selected from —X¹—, —X²—C₁₋₆ alkylene-X²—C₁₋₆ alkylene-, —X²—C₂₋₆alkenylene-X²—, and —X²—C₂₋₆ alkynylene-X²—, each of which is optionallysubstituted on alkylene, alkenylene or alkynylene with one or more R¹²;

L² is selected from —X²—, —X²—C₁₋₆ alkylene-X²—, —X²—C₂₋₆alkenylene-X²—, and

—X²—C₂₋₆ alkynylene-X²—, each of which is optionally substituted onalkylene, alkenylene or alkynylene with one or more R¹²;

X¹ is selected from —S—*, —N(R¹⁰)—*, —C(O)O—*, —OC(O)—*, —OC(O)O—*,—C(O)N(R¹⁰)C(O)—*, —C(O)N(R¹⁰)C(O)N(R¹⁰)*, —N(R¹⁰)C(O)—*, —CR¹⁰₂N(R¹⁰)C(O)—*, —N(R¹⁰)C(O)N(R¹⁰)—*, —N(R¹⁰)C(O)O—*, —OC(O)N(R¹⁰)—*,—C(NR¹⁰)—*, —N(R¹⁰)C(NR¹⁰)—*, —C(NR¹⁰)N(R¹⁰)—*, —N(R¹⁰)C(NR¹⁰)N(R¹⁰)—*,—S(O)₂—*, —OS(O)—*, —S(O)O—*, —S(O), —OS(O)₂—*, —S(O)₂O*,—N(R¹⁰)S(O)₂—*, —S(O)₂N(R¹⁰)—*, —N(R¹⁰)S(O)—*, —S(O)N(R¹⁰)—*,—N(R¹⁰)S(O)₂N(R¹⁰)—*, and —N(R¹⁰)S(O)N(R¹⁰)—*, wherein * representswhere X¹ is bound to R³;

X² is independently selected at each occurrence from —O—, —S—, —N(R¹⁰)—,—C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R¹⁰)—, —C(O)N(R¹⁰)C(O)—,—C(O)N(R¹⁰)C(O)N(R¹⁰), —N(R¹⁰)C(O)—, —N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)O—,—OC(O)N(R¹⁰)—, —C(NR¹⁰)—, —N(R¹⁰)C(NR¹⁰)—, —C(NR¹⁰)N(R¹⁰)—,—N(R¹⁰)C(NR¹⁰)N(R¹⁰)—, —S(O)₂—, —OS(O)—, —S(O)O—, —S(O), —OS(O)₂—,—S(O)₂O, —N(R¹⁰)S(O)₂—, —S(O)₂N(R¹⁰)—, —N(R¹⁰)S(O)—, —S(O)N(R¹⁰)—,—N(R¹⁰)S(O)₂N(R¹⁰)—, and —N(R¹⁰)S(O)N(R¹⁰)—;

R¹ and R² are independently selected from hydrogen; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl, each of which is optionally substituted withone or more substituents independently selected from halogen, —OR¹⁰,—SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN;

R³ is selected from optionally substituted C₃₋₁₂ carbocycle, andoptionally substituted 3- to 12-membered heterocycle, whereinsubstituents on R³ are independently selected at each occurrence from:halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and—CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O,═S, ═N(R¹⁰), —CN, C₁₋₁₂ carbocycle, and 3- to 12-membered heterocycle;and C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, wherein eachC₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle in R³ is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN,C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl;

R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle in R⁴ is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, Cue alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl;

R¹⁰ is independently selected at each occurrence from: hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, C₁₋₂₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, 3- to 12-membered heterocycle, and haloalkyl; and

R¹² is independently selected at each occurrence from halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀carbocycle and 3- to 10-membered heterocycle; and C₃₋₁₀ carbocycle and3- to 10-membered heterocycle, wherein each C₃₋₁₀ carbocycle and 3- to10-membered heterocycle in R¹² is optionally substituted with one ormore substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl; and

wherein any substitutable carbon on the benzazepine core is optionallysubstituted by a substituent independently selected from R¹² or twosubstituents on a single carbon atom combine to form a 3- to 7-memberedcarbocycle.

In some embodiments, the compound of Formula (IA) is represented byFormula (IB):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R²⁰, R²¹, R²², and R²³ are independently selected from hydrogen,        halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,        —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl; and    -   R²⁴ and R²⁵ are independently selected from hydrogen, halogen,        —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,        —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl; or R²⁴ and R²⁵ taken together        form an optionally substituted saturated C₃₋₇ carbocycle.

In some embodiments, R²⁰, R²¹, R²², and R²³ are independently selectedfrom hydrogen, halogen, —OH, —NO₂, —CN, and C₁₋₁₀ alkyl. In certainembodiments, R²⁰, R²¹, R²², and R²³ are each hydrogen.

In some embodiments, R²⁴ and R²⁵ are independently selected fromhydrogen, halogen, —OH, —NO₂, —CN, and C₁₋₁₀ alkyl, or R²⁴ and R²⁵ takentogether form an optionally substituted saturated C₃₋₇ carbocycle. Insome embodiments, R²⁴ and R²⁵ are each hydrogen. In some embodiments,R²⁴ and R²⁵ taken together form an optionally substituted saturated C₃₋₅carbocycle.

In some embodiments, R¹ is hydrogen. In some embodiments, R² ishydrogen.

In some embodiments, L¹ is selected from —N(R¹⁰)C(O)—*, —S(O)₂N(R¹⁰)—*,—CR¹⁰2N(R¹⁰)C(O)—* and —X²—C₁₋₆ alkylene-X²—C₁₋₆ alkylene-. In someembodiments, L¹ is selected from —N(R¹⁰)C(O)—*. In certain embodiments,R¹⁰ of —N(R¹⁰)C(O)—* is selected from hydrogen and C₁₋₆ alkyl. Forexample, L¹ may be —NHC(O)—*. In some embodiments, L¹ is selected from—S(O)₂N(R¹⁰)—*. In certain embodiments, R¹⁰ of —S(O)₂N(R¹⁰)—* isselected from hydrogen and C₁₋₆ alkyl. For example, L¹ is —S(O)₂NH—*. Insome embodiments, L¹ is —CR¹⁰ ₂N(R¹⁰)C(O)—*. In certain embodiments, L¹is selected from —CH₂N(H)C(O)—* and —CH(CH₃)N(H)C(O)—*.

In some embodiments, R³ is selected from optionally substituted C₃₋₁₂carbocycle, and optionally substituted 3- to 12-membered heterocycle,wherein substituents on R³ are independently selected at each occurrencefrom: halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl. In certain embodiments, R³ is selected fromoptionally substituted C₃₋₁₂ carbocycle, and optionally substituted 3-to 12-membered heterocycle, wherein substituents on R³ are independentlyselected at each occurrence from: halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle.

In some embodiments, R³ is selected from an optionally substituted aryland an optionally substituted heteroaryl. In some embodiments, R³ is anoptionally substituted heteroaryl. R³ may be an optionally substitutedheteroaryl substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl. In certain embodiments, R³ is selected from an optionallysubstituted 6-membered heteroaryl. For example, R³ may be an optionallysubstituted pyridine. In some embodiments, R³ is an optionallysubstituted aryl. In certain embodiments, R³ is an optionallysubstituted aryl substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl. R³ may be an optionally substituted phenyl. In certainembodiments, R³ is selected from pyridine, phenyl,tetrahydronaphthalene, tetrahydroquinoline, tetrahydroisoquinoline,indane, cyclopropylbenzene, cyclopentapyridine, anddihydrobenzoxaborole, any one of which is optionally substituted. R³ maybe selected from:

any one of which is optionally substituted. For example, R³ may beselected from:

In some embodiments, L² is selected from —C(O)—, and —C(O)NR¹⁰—. Incertain embodiments, L² is —C(O)—. In certain embodiments, L² isselected from —C(O)NR¹⁰—. R¹⁰ of —C(O)NR¹⁰— may be selected fromhydrogen and C₁₋₆ alkyl. For example, L² may be —C(O)NH—.

In some embodiments, R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OR¹⁰,—SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl.

In some embodiments, R⁴ is selected from: —OR¹⁰, —N(R¹⁰)₂, —C(O)N(R¹⁰)₂,—C(O)R¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle. In some embodiments, R⁴ is selected from:—OR¹⁰, and —N(R¹⁰)₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰,—C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl. In certain embodiments, R⁴ is—N(R¹⁰)₂. R¹⁰ of —N(R¹⁰)₂ may be independently selected at eachoccurrence from optionally substituted C₁₋₆ alkyl. In certainembodiments, R¹⁰ of —N(R¹⁰)₂ is independently selected at eachoccurrence from methyl, ethyl, propyl, and butyl, any one of which isoptionally substituted. For example, R⁴ may be

In certain embodiments, L²-R⁴ is

In some embodiments, R¹² is independently selected at each occurrencefrom halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)_(2J)—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S,═N(R¹⁰), —CN, C₃₋₁₀ carbocycle and 3- to 10-membered heterocycle; andC₃₋₁₀ carbocycle and 3- to 10-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl. In some embodiments, R¹² is independently selected at eachoccurrence from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂,—OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₃₋₁₀ carbocycle and 3- to 10-membered heterocycle.

In some embodiments, the compound is selected from:

and a salt of any one thereof.

In some aspects, the present disclosure provides a compound representedby the structure of Formula (IVA):

or a pharmaceutically acceptable salt thereof, wherein:

represents an optional double bond;

L¹² is selected from —X³—, —X³—C₁₋₆alkylene-X³—, —X³—C₂₋₆alkenylene-X³—, and —X³—C₂₋₆ alkynylene-X³—, each of which is optionallysubstituted on alkylene, alkenylene, or alkynylene with one or moresubstituents independently selected from R¹²;

L²² is independently selected from —X⁴—, —X⁴—C₁₋₆alkylene-X⁴—, —X⁴—C₂₋₆alkenylene-X⁴—, and —X⁴—C₂₋₆ alkynylene-X⁴—, each of which is optionallysubstituted on alkylene, alkenylene, or alkynylene with one or moresubstituents independently selected from R¹⁰;

X³ and X⁴ are independently selected at each occurrence from a bond,—O—, —S—, —N(R¹⁰)—, —C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R¹⁰)—,—C(O)N(R¹⁰)C(O)—, —C(O)N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)—,—N(R¹⁰)C(O)N(R¹⁰)—, —N(R¹⁰)C(O)O—, —OC(O)N(R¹⁰)—, —C(NR¹⁰)—,—N(R¹⁰)C(NR¹⁰)—, —C(NR¹⁰)N(R¹⁰)—, —N(R¹⁰)C(NR¹⁰)N(R¹⁰)—, —S(O)₂—,—OS(O)—, —S(O)O—, —S(O)—, —OS(O)₂—, —S(O)₂O—, —N(R¹⁰)S(O)₂—,—S(O)₂N(R¹⁰)—, —N(R¹⁰)S(O)—, —S(O)N(R¹⁰)—, —N(R¹⁰)S(O)₂N(R¹⁰)—, and—N(R¹⁰)S(O)N(R¹⁰)—;

R¹ and R² are independently selected from L³, and hydrogen; and C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl, each of which is optionallybound to L³ and each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and —CN;

R⁴ and R⁸ are independently selected from: —OR¹⁰, —N(R¹⁰)₂,—C(O)N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, and —S(O)₂R¹⁰; C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which is optionally bound to L³and each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₂ carbocycle, and3- to 12-membered heterocycle; and C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, wherein each C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle in R⁴ and R⁸ is optionally bound to L³ and eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂,—N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰,—OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl;

R¹⁰ is independently selected at each occurrence from L³, hydrogen,—NH₂, —C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, 3- to 12-membered heterocycle, and haloalkyl;

L³ is a linker moiety, wherein there is at least one occurrence of L³;and

R¹² is independently selected at each occurrence from halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰,—OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O,═S, ═N(R¹⁰), and —CN; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —C(O)R¹⁰,—C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰—C(O)OR¹⁰, —OC(O)R¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₃₋₁₀carbocycle and 3- to 10-membered heterocycle; and C₃₋₁₀ carbocycle and3- to 10-membered heterocycle, wherein each C₃₋₁₀ carbocycle and 3- to10-membered heterocycle in R¹² is optionally substituted with one ormore substituents independently selected from halogen, —OR¹⁰, —SR¹⁰,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—S(O)R¹⁰, S(O)₂R¹⁰, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —NO₂, ═O, ═S, ═N(R¹⁰),—CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl;

wherein any substitutable carbon on the benzazepine core is optionallysubstituted by a substituent independently selected from R¹² or twosubstituents on a single carbon atom combine to form a 3- to 7-memberedcarbocycle.

In some embodiments, the compound of Formula (IVA) is represented byFormula (IVB):

or a pharmaceutically acceptable salt thereof, wherein:

R²⁰, R²¹, R²², and R²³ are independently selected from hydrogen,halogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl; and

R²⁴, and R²⁵ are independently selected from hydrogen, halogen, —OR¹⁰,—SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰,—NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; or R²⁴ and R²⁵ taken together form an optionally substitutedsaturated C₃₋₇ carbocycle.

In some embodiments, R¹ is L³. In some embodiments, R² is L³.

In some embodiments, L¹² is —C(O)N(R¹⁰)—. In some embodiments, R¹⁰ of—C(O)N(R¹⁰)— is selected from hydrogen, C₁₋₆ alkyl, and L³. For example,L¹² may be —C(O)NH—.

In some embodiments, R⁸ is an optionally substituted 5- or 6-memberedheteroaryl. R⁸ may be an optionally substituted 5- or 6-memberedheteroaryl, bound to L³. In some embodiments, R⁸ is an optionallysubstituted pyridine, bound to L³.

In some embodiments, L²² is selected from —C(O)—, and —C(O)NR¹⁰—. Incertain embodiments, L²² is —C(O)—. In certain embodiments, L²² is—C(O)NR¹⁰—. R¹⁰ of —C(O)NR¹⁰— may be selected from hydrogen, C₁₋₆ alkyl,and -L³. For example, L²² may be —C(O)NH—.

In some embodiments, R⁴ is selected from: —OR¹⁰, and —N(R¹⁰)₂; and C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂ carbocycle, 3- to 12-memberedheterocycle, aryl, and heteroaryl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —N(R¹⁰)₂, —S(O)R¹⁰, —S(O)₂R¹⁰, —C(O)R¹⁰,—C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl and each of which is further optionally boundto L³. In some embodiments, R⁴ is —N(R¹⁰)₂ and R¹⁰ of —N(R¹⁰)₂ isselected from L³ and hydrogen, and wherein at least one R¹⁰ of —N(R¹⁰)₂is L³.

In some aspects, the compound of Formula (IVB) is a compound of Formula(IVC):

or a pharmaceutically acceptable salt thereof,

wherein:

R¹ and R² are hydrogen;

L²² is —C(O)—;

R⁴—N(R¹⁰)₂;

R¹⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅,—NHC(O)OCH₂C₆H₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₂carbocycle, 3- to 12-membered heterocycle, and haloalkyl;

L¹² is —C(O)N(R¹⁰)—*, wherein * represents where L¹² is bound to R⁸;

R⁸ is an optionally substituted fused 5-5, fused 5-6, or fused 6-6bicyclic heterocycle bound to linker moiety, L³, and wherein optionalsubstituents are independently selected at each occurrence from:

halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), and—CN;

C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰, —N(R¹⁰)C(O)N(R¹⁰)₂,—N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O, ═S, ═N(R¹⁰), —CN,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle; and

C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each of which isoptionally substituted with one or more substituents independentlyselected from halogen, —OR¹⁰, —SR¹⁰, —C(O)N(R¹⁰)₂, —N(R¹⁰)C(O)R¹⁰,—N(R¹⁰)C(O)N(R¹⁰)₂, —N(R¹⁰)₂, —C(O)R¹⁰, —C(O)OR¹⁰, —OC(O)R¹⁰, —NO₂, ═O,═S, ═N(R¹⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl.

In certain embodiments: R¹⁰ of —N(R¹⁰)₂ is independently selected ateach occurrence from methyl, ethyl, propyl, and butyl, any one of whichis optionally substituted. In certain embodiments, R¹⁰ of —C(O)N(R¹⁰)—*is hydrogen.

In some embodiments, the compound is further covalently bound to alinker, L³. In some embodiments, L³ is a noncleavable linker. In someembodiments, L³ is a cleavable linker. L³ may be cleavable by alysosomal enzyme. In some embodiments, the compound is covalentlyattached to an antibody or antigen binding fragment thereof.

In some embodiments, L³ is represented by the formula:

wherein:

L⁴ represents the C-terminus of the peptide and L⁵ is selected from abond, alkylene and heteroalkylene, wherein L⁵ is optionally substitutedwith one or more groups independently selected from R³², and RX is areactive moiety; and

R³² is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OH, —CN, —O-alkyl,—SH, ═O, ═S, —NH₂, —NO₂.

In some embodiments, RX comprises a leaving group. In some embodiments,RX comprises a maleimide. In some embodiments, L³ is further covalentlybound to an antibody or antigen binding fragment thereof.

In some embodiments, L³ is represented by the formula:

wherein

L⁴ represents the C-terminal of the peptide and

L⁵ is selected from a bond, alkylene and heteroalkylene,

-   -   wherein L⁵ is optionally substituted with one or more groups        independently selected from R³²;

RX* comprises a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of an antibody or antigen binding fragmentthereof,

-   -   wherein

-   -    on RX* represents the point of attachment to the residue of the        antibody or antigen binding fragment thereof; and,

R³² is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OH, —CN, —O-alkyl,—SH, ═O, ═S, —NH₂, —NO₂. In some embodiments, the peptide of L³comprises Val-Cit or Val-Ala.

In some aspects, the present disclosure provides a compound or saltselected from:

and a salt of any one thereof.

In some aspects, the present disclosure provides a compound or saltselected from:

and a salt of any one thereof,

wherein the RX* is a bond, a succinimide moiety, or a hydrolyzedsuccinimide moiety bound to a residue of an antibody or antigen bindingfragment thereof,

wherein

on RX* represents the point of attachment to the residue of the antibodyor antigen binding fragment thereof.

In some embodiments, L³ is represented by the formula:

wherein RX comprises a reactive moiety, and n=0-9. In some embodiments,RX comprises a leaving group. In some embodiments, RX comprises amaleimide. In some embodiments. L³ is represented as follows:

wherein RX* comprises a bond, a succinimide moiety, or a hydrolyzedsuccinimide moiety bound to a residue of an antibody or antigen bindingfragment, wherein

on RX* represents the point of attachment to the residue of the antibodyor antigen binding fragment thereof, and n=0-9.

In some aspects, the present disclosure provides a compound or saltselected from:

and a salt of any one thereof.

In some aspects, the present disclosure provides a compound or saltselected from:

and a salt of any one thereof, wherein the RX* comprises a bond, asuccinimide moiety, or a hydrolyzed succinimide moiety bound to aresidue of an antibody or antigen binding fragment thereof, wherein

on RX* represents the point of attachment to the residue of the antibodyor antigen binding fragment thereof.

In some embodiments, RX* comprises a succinamide moiety and is bound toa cysteine residue of an antibody or antigen binding fragment thereof.In some embodiments, RX* comprises a hydrolyzed succinamide moiety andis bound to a cysteine residue of an antibody or antigen bindingfragment thereof.

In some aspects, the present disclosure provides a conjugate representedby the formula:

wherein Antibody is an anti-Nectin-4 antibody or antigen bindingfragment thereof disclosed herein, D is a Category A compound or saltdisclosed herein, and L³ is a linker moiety.

In some aspects, the present disclosure provides a conjugate representedby the formula:

wherein Antibody is an anti-Nectin-4 antibody or antigen bindingfragment thereof disclosed herein, and D-L³ is a Category A compound orsalt disclosed herein.

In some aspects, the present disclosure provides a pharmaceuticalcomposition, comprising the conjugate disclosed herein and at least onepharmaceutically acceptable excipient.

In some embodiments, the average DAR of the conjugate is from about 2 toabout 8, or about 1 to about 3, or about 3 to about 5.

Examples of TLR8 agonist compounds according to Category A are providedin Table 1a and their stereoisomers. It is understood that a compound isprovided in Table 1a, salts of that compound are envisioned by Table 1a.

TABLE 1a Compounds 1.1-1.69 Compound Structure and IUPAC 1.1 

2-amino-N4,N4-dipropyl-N8-(1,2,3,4-tetrahydroquinolin-7-yl)-3H-benzo[b]azepine-4,8- dicarboxamide 1.2 

N⁸-(3-acetylphenyl)-2-amino-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.3 

2-amino-N⁴,N⁴-dipropyl-N⁸-(pyridin-3-ylmethyl)-3H-benzo[b]azepine-4,8-dicarboxamide 1.4 

2-amino-N⁸-(8-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.5 

2-amino-N⁸-(5-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.6 

2-amino-N⁸-(3-(hydrazinecarbonyl)phenyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.7 

2-amino-N⁸-(8-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.8 

2-amino-N⁸-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.9 

2-amino-N⁸-(4-(3-hydroxypiperidin-1-yl)phenyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.10

2-amino-N⁸-(4-(4-hydroxypiperidin-1-yl)phenyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.11

N⁸-(4-(4-acetylpiperidin-1-yl)phenyl)-2-amino-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.12

2-amino-N⁴,N⁴-dipropyl-N⁸-(1,2,3,4-tetrahydroquinolin-6-yl)-3H-benzo[b]azepine-4,8- dicarboxamide 1.13

2-amino-N⁴,N⁴-dipropyl-N⁸-(1,2,3,4-tetrahydroisoquinolin-6-yl)-3H-benzo[b]azepine- 4,8-dicarboxamide 1.14

2-amino-N⁴,N⁴-dipropyl-N⁸-(1,2,3,4-tetrahydro-isoquinolin-7-yl)-3H-benzo[b]azepine-4,8- dicarboxamide trifluoroacetatesalt 1.15

benzyl (S)-(1-(((5-(2-amino-4-(dipropyl-carbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin-3-yl)methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 1.16

benzyl (S)-(1-(((5-(2-amino-4-(dipropyl-carbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin-3-yl)methyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate 1.17

benzyl (S)-2-(((5-(2-amino-4-(dipropyl- carbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin-3- yl)methyl)carbamoyl)pyrrolidine-1-carboxylate1.18

methyl (3R,4S)-4-(3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)phenyl)-1-benzylpyrrolidine-3- carboxylate 1.19

methyl (3R,4S)-4-(4-(2-amino-4-(dipropyl-carbamoyl)-3H-benzo[b]azepine-8-carboxamido)phenyl)-1-benzylpyrrolidine-3- carboxylate 1.20

benzyl ((6-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-3- yl)methyl)carbamate 1.21

(S)-2-amino-N⁸-(1-phenylethyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.22

(R)-2-amino-N⁸-(1-phenylethyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.23

2-amino-N⁸-(2,3-dihydro-1H-inden-1-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.24

2-amino-N,N-dipropyl-8-(1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3H-benzo[b]azepine-4-carboxamide 1.25

N⁸-(4-acetylphenyl)-2-amino-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.26

benzyl (2-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)ethyl)carbamate 1.27

benzyl (2-(3-(2-amino-4-(dipropylcarbamoyl)- 3H-benzo[b]azepine-8-carboxamido)benzamido)ethyl)carbamate 1.28

2-amino-N⁸-((1S,2R)-2-phenylcyclopropyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.29

benzyl 6-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate 1.30

benzyl 7-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate 1.31

2-amino-N⁸-(3-((3- phenylpropanamido)methyl)phenyl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.32

2-amino-N⁸-(5-((3-benzylureido)methyl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.33

2-amino-N⁴,N⁴-dipropyl-N⁸-(5-((1,2,3,4- tetrahydroquinoline-2-carboxamido)methyl)pyridin-3-yl)-3H- benzo[b]azepine-4,8-dicarboxamide1.34

2-amino-N⁴,N⁴-dipropyl-N⁸-(5-((1,2,3,4-tetrahydroisoquinoline-3-carboxamido)-methyl)pyridin-3-yl)-3H-benzo[b]azepine-4,8- dicarboxamide 1.35

(S)-2-amino-N⁸-(5-((2-amino-3-phenylpropanamido)methyl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.36

(R)-2-amino-N⁸-(5-((2-amino-3-phenyl-propanamido)-methyl)pyridine-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.37

Phenyl ((5-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin-3- yl)methyl)carbamate 1.38

2-amino-N⁸-(5-((3-amino-3-phenyl-propanamido)methyl)-pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.39

2-amino-N⁸-(5-amino-5,6,7,8-tetrahydro-quinolin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo- [b]azepine-4,8-dicarbox-amide1.40

Benzyl (3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-5,6,7,8-tetrahydroquinolin-5-yl)carbamate 1.41

2-amino-N⁸-(5-amino-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.42

Benzyl (3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)carbamate 1.43

N⁸-(6-acetylpyridin-3-yl)-2-amino-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.44

2-amino-N⁸-(3-amino-2,3-dihydro-1H-inden-5-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.45

Benzyl (6-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-2,3-dihydro- 1H-inden-1-yl)carbamate 1.46

2-amino-N⁸-(5-((4- phenylbutanamido)methyl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.47

2-amino-N⁸-((1-hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-3-yl)methyl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.48

2-amino-N⁸-(6-benzyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-N⁴,N⁴-dipropyl-3H- benzo[b]azepine-4,8-dicarboxamide1.49

benzyl (3-((2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)methyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6- yl)carbamate 1.50

2-amino-N⁴,N⁴-dipropyl-N⁸-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-3H-benzo[b]azepine-4,8- dicarboxamide 1.51

(S)-2-amino-N⁸-(5-((2-amino-3-methylbutanamido)methyl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.52

benzyl (3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-5,6,7,8-tetrahydroquinolin-7-yl)carbamate 1.53

benzyl (3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-6,7-dihydro-5H-cyclopenta[b]pyridin-6-yl)carbamate 1.54

benzyl 3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 1.55

benzyl (1-(5-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin-2- yl)piperidin-3-yl)carbamate1.56

2-amino-N⁸-(6-(3-aminopiperidin-1-yl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.57

2-amino-N⁸-(6-(4-aminopiperidin-1-yl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.58

2-amino-N⁴,N⁴-dipropyl-N⁸-(5-(pyrrolidin-3-yl)pyridin-3-yl)-3H-benzo[b]azepine-4,8- dicarboxamide 1.59

benzyl (2-(4-((3-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methyl)benzamido)ethyl)carbamate 1.60

2-amino-N⁸-(6-(4-((2- aminoethyl)carbamoyl)benzyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.61

2-amino-N⁸-(6-(4-((2- aminoethyl)carbamoyl)piperidin-1-yl)pyridin-3-yl)-N⁴,N⁴-dipropyl-3H-benzo[b]azepine-4,8- dicarboxamide 1.62

2-amino-8-(nicotinamido)-N,N-dipropyl-3H- benzo[b]azepine-4-carboxamide1.63

2-amino-N,N-dipropyl-8-(N-(pyridin-3-yl)sulfamoyl)-3H-benzo[b]azepine-4- carboxamide 1.64

2-amino-N8-(5-((2- aminoacetamido)methyl)pyridin-3-yl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.65

2-amino-7-methoxy-N4,N4-dipropyl-N8-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-3H- benzo[b]azepine-4,8-dicarboxamide1.66

2-amino-7-fluoro-N4,N4-dipropyl-N8-(5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-3H- benzo[b]azepine-4,8-dicarboxamide1.67

2-amino-N8-(6-(4-((3-amino-2,2-difluoropropyl)carbamoyl)benzyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide 1.68

1.69

Compounds of Category B, TLR7 Agonists

In some aspects, the present disclosure provides a compound representedby the structure of Formula (IA):

or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, R⁴, and R⁵ are independently selected from hydrogen; andC₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN; or R³and R¹¹ taken together form a 5- to 10-membered heterocycle optionallysubstituted with one or more substituents independently selected fromhalogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN;

R⁶ is selected from halogen, —OR²⁰, —N(R²⁰)₂, —C(O)N(R²⁰)₂, —C(O)R²⁰,—C(O)OR²⁰, —S(O)R²⁰, and —S(O)₂R²⁰; and C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected at each occurrence fromhydrogen and halogen; and C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl,each of which is optionally substituted with one or more substituentsindependently selected from halogen;

R¹¹ and R¹² are independently selected from hydrogen, halogen, —OR²⁰,—SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, and —CN; and C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; or R¹¹ and R¹² taken together form a C₃₋₆carbocycle optionally substituted with one or more substituentsindependently selected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂,—N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O,═S, ═N(R²⁰), and —CN;

R¹³ and R¹⁴ are independently selected at each occurrence from hydrogen,halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, and —CN; Cue alkyl, C₂₋₆ alkenyl,and C₂₋₆ alkynyl, each of which is optionally substituted with one ormore substituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle and 3- to 12-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl;

R¹⁵ is independently selected at each occurrence from halogen, —OR²⁰,—SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S, —C₁₋₆alkyl, —C₁₋₆ haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle;

R¹⁶ is selected from hydrogen; and C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S,C₁₋₆alkyl, —C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle;

R²⁰ is independently selected at each occurrence from hydrogen; and C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OH, —CN,—NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆ alkyl,—C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle;

X¹ is O, S, or NR¹⁶;

X² is C(O) or S(O)₂;

n is 1, 2, or 3;

x is 1, 2, or 3;

w is 0, 1, 2, 3, or 4; and

z is 0, 1, or 2.

In certain embodiments, for a compound of Formula (IA), wherein X¹ is O.In certain embodiments, for a compound of Formula (IA), n is 2. Incertain embodiments, for a compound of Formula (IA), x is 2. In certainembodiments, for a compound of Formula (IA), z is 0. In certainembodiments, for a compound of Formula (IA), z is 1.

In certain embodiments, a compound of Formula (IA) is represented byFormula (IB):

or a pharmaceutically acceptable salt thereof, wherein R^(7′), R^(7″),R^(8′), R^(8″), R^(9′), R^(9″), R^(10′), and R^(10″) are independentlyselected at each occurrence from hydrogen and halogen; and C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆alkynyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen.

In certain embodiments, a compound of Formula (IA) is represented byFormula (IC):

or a pharmaceutically acceptable salt thereof, wherein R^(7′), R^(7″),R^(8′), R^(8″), R^(9′), R^(9″), R^(10′), and R^(10″) are independentlyselected at each occurrence from hydrogen and halogen; and C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R¹, R², R³, R⁴, and R⁵ are independently selectedfrom hydrogen and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R¹ and R² are independently selected from hydrogenand C₁₋₆ alkyl. In certain embodiments, for a compound or salt of anyone of Formulas (IA), (IB), or (IC), R¹ and R² are each hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R³ is selected from hydrogen and C₁₋₆ alkyloptionally substituted with one or more halogens.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R³ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R⁴ is selected from hydrogen and C₁₋₆ alkyloptionally substituted with one or more halogens.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R⁴ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R⁵ is selected from hydrogen and C₁₋₆ alkyloptionally substituted with one or more substituents independentlyselected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰,—S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and—CN. In certain embodiments, for a compound or salt of any one ofFormulas (IA), (IB), or (IC), R⁵ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R⁶ is selected from halogen, —OR²⁰, and —N(R²⁰)₂;and C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN; and

R²⁰ is independently selected at each occurrence from hydrogen; and C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OH, —CN,—NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆ alkyl,—C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC),

R⁶ is C₁₋₆ alkyl optionally substituted with one or more substituentsindependently selected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂,—N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰; and

R²⁰ is independently selected at each occurrence from hydrogen; C₁₋₆alkyl, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S,—C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆ alkyl, —C₁₋₆haloalkyl, —O—C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R⁶ is C₁₋₆ alkyl substituted with —OR²⁰, and R²⁰ isselected from hydrogen and C₁₋₆ alkyl optionally substituted with one ormore substituents independently selected from halogen, —OH, and —NH₂.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R^(7′), R^(7″), R^(8′), R^(8″), R^(9′), R^(9″),R^(10′), and R^(10∝) are independently selected at each occurrence fromhydrogen and halogen; and C₁₋₆ alkyl, optionally substituted with one ormore substituents independently selected from halogen.

In certain embodiments, for a compound or salt of any one of Formulas(IB) or (IC), wherein R^(7′) and R^(8′) are each hydrogen. In certainembodiments, for a compound or salt of any one of Formulas (IB) or (IC),wherein R^(7″) and R^(8″) are each C₁₋₆ alkyl. In certain embodiments,for a compound or salt of any one of Formulas (IB) or (IC), R^(7″) andR^(8″) are each methyl.

In certain embodiments, for a compound or salt of any one of Formulas(IB) or (IC), R^(9′), R^(9″), R^(10′), and R^(10″) are independentlyselected at each occurrence from hydrogen and C₁₋₆ alkyl.

In certain embodiments, for a compound or salt of any one of Formulas(IB) or (IC), R^(9′), R^(9″), R^(10′), and R^(10″) are each hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R¹¹ and R¹² are independently selected fromhydrogen, halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰,—C(O)OR²⁰, —OC(O)R²⁰; and C₁₋₆ alkyl, optionally substituted with one ormore substituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA) or (IC), R¹³ and R¹⁴ are independently selected from hydrogen,halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰; and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R³ and R¹¹ taken together form an optionallysubstituted 5- to 6-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), R¹¹ and R¹² taken together form an optionallysubstituted C₃₋₆ carbocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), X² is C(O).

In certain embodiments, the compound is represented by:

or a pharmaceutically acceptable salt of any one thereof.

In certain aspects, the disclosure provides a pharmaceutical compositionof a compound or pharmaceutically acceptable salt of any one of Formulas(IA), (IB), or (IC), and a pharmaceutically acceptable excipient.

In certain embodiments, for a compound or salt of any one of Formulas(IA), (IB), or (IC), the compound or salt is further covalently bound toa linker, L³.

In certain aspects the disclosure provides a compound represented byFormula (HA):

or a pharmaceutically acceptable salt thereof, wherein:

R² and R⁴ are independently selected from hydrogen; and C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl, each of which is optionally substituted withone or more substituents independently selected from halogen, —OR²⁰,—SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R^(2c), —NO₂, ═O, ═S, ═N(R²⁰), and —CN;

R²¹, R²³, and R²⁵ are independently selected from hydrogen; C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen,—OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰,—C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN; and L³; or R²³ andR¹¹ taken together form a 5- to 10-membered heterocycle optionallysubstituted with one or more substituents independently selected fromhalogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN; andwherein one of R²¹, R²³, and R²⁵ is L³;

R⁶ is selected from halogen, —OR²⁰, —N(R²⁰)₂, —C(O)N(R²⁰)₂, —C(O)R²⁰,—C(O)OR²⁰, —S(O)R²⁰, and —S(O)₂R²⁰; and C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected at each occurrence fromhydrogen and halogen; and C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl,each of which is optionally substituted with one or more substituentsindependently selected from halogen;

R¹¹ and R¹² are independently selected from hydrogen, halogen, —OR²⁰,—SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, and —CN; and C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; or R¹¹ and R¹² taken together form a C₃₋₆carbocycle optionally substituted with one or more substituentsindependently selected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂,—N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁹, —OC(O)R²⁰, —NO₂, ═O,═S, ═N(R²⁰), and —CN;

R¹³ and R¹⁴ are independently selected at each occurrence from hydrogen,halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰,—C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle; and C₃₋₁₂ carbocycle and 3- to 12-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, andC₂₋₆alkynyl;

R¹⁵ is independently selected at each occurrence from halogen, —OR²⁰,—SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S,—C₁₋₆alkyl, —C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl,C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle;

R¹⁶ is selected from hydrogen; and C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂ carbocycle, and 3- to 12-membered heterocycle, each ofwhich is optionally substituted with one or more substituentsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, ═O, ═S, C₁₋₆alkyl, —C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle;

R²⁰ is independently selected at each occurrence from hydrogen; C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OH, —CN,—NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆ alkyl,—C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle;

L³ is a linker;

X¹ is O, S, or NR¹⁶;

X² is C(O) or S(O)₂;

n is 1, 2, or 3;

x is 1, 2, or 3;

w is 0, 1, 2, 3, or 4; and

z is 0, 1, or 2.

In certain embodiments, for a compound or salt of Formula (IIA), X¹ isO. In certain embodiments, for a compound or salt of Formula (IIA), n is2. In certain embodiments, for a compound or salt of Formula (IIA), x is2. In certain embodiments, for a compound or salt of Formula (IIA), z is0. In certain embodiments, for a compound or salt of Formula (IIA), z is1.

In certain embodiments, the compound of Formula (IIA) is represented by(IIB) or (IIC):

or a pharmaceutically acceptable salt thereof, wherein R^(7′), R^(7″),R^(8′), R^(8″), R^(9′), R^(9″), R^(10′), and R^(10″) are independentlyselected at each occurrence from hydrogen and halogen; and C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R² and R⁴ are independently selected fromhydrogen and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R² and R⁴ are independently selected fromhydrogen and C₁₋₆ alkyl. In certain embodiments, for a compound or saltof any one of Formulas (IIA), (IIB), or (IIC), R² and R⁴ are eachhydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²³ is selected from hydrogen and C₁₋₆ alkyloptionally substituted with one or more halogens. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), R²³ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²¹ is selected from hydrogen and C₁₋₆ alkyloptionally substituted with one or more halogens. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), R²¹ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²¹ is L³.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²⁵ is selected from hydrogen and C₁₋₆ alkyl,optionally substituted with one or more substituents independentlyselected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰,—S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and—CN. In certain embodiments, for a compound or salt of any one ofFormulas (IIA), (IIB), or (IIC), R²⁵ is hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²⁵ is L³.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC),

R⁶ is selected from halogen, —OR²⁰, and —N(R²⁰)₂; and C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, each of which is optionally substituted with oneor more substituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰,—OC(O)R²⁰, —NO₂, ═O, ═S, ═N(R²⁰), and —CN; and

R²⁰ is independently selected at each occurrence from hydrogen; and C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle, each of which is optionally substituted withone or more substituents independently selected from halogen, —OH, —CN,—NO₂, —NH₂, ═O, ═S, —C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆ alkyl, —C₁₋₆haloalkyl, —O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₂ carbocycle,and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC),

R⁶ is C₁₋₆ alkyl optionally substituted with one or more substituentsindependently selected from halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂,—N(R²⁰)₂, —S(O)R²⁰, —S(O)₂R²⁰, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰; and

R²⁰ is independently selected at each occurrence from hydrogen, —NH₂,—C(O)OCH₂C₆H₅; C₁₋₆ alkyl, C₃₋₁₂ carbocycle, and 3- to 12-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OH, —CN, —NO₂, —NH₂,═O, ═S, —C(O)OCH₂C₆H₅, —NHC(O)OCH₂C₆H₅, C₁₋₆alkyl, —C₁₋₆ haloalkyl,—O—C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₂ carbocycle, and 3- to12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC),

R⁶ is C₁₋₆ alkyl substituted with —OR²⁰, and

R²⁰ is selected from hydrogen and C₁₋₆ alkyl, which is optionallysubstituted with one or more substituents independently selected fromhalogen, —OH, and —NH₂.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(7′), R^(7″), R^(8′), R^(8″), R^(9′), R^(9″), R^(10′),and R^(10″) are independently selected at each occurrence from hydrogenand halogen; and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(7′) and R^(8′) are hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(7″) and R^(8″) are C₁₋₆ alkyl.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(7″) and R^(8″) are methyl.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(9′), R^(9″), R^(10′), and R^(10″) are independentlyselected at each occurrence from hydrogen and C₁₋₆ alkyl.

In certain embodiments, for a compound or salt of any one of Formulas(IIB) or (IIC), R^(9′), R^(9″), R^(10′), and R^(10″) are each hydrogen.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R¹¹ and R¹² are independently selected fromhydrogen, halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰,—C(O)OR²⁰, and —OC(O)R²⁰; and C₁₋₆ alkyl optionally substituted with oneor more substituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA) or (IIC), R¹³ and R¹⁴ are independently selected from hydrogen,halogen, —OR²⁰, —SR²⁰, —C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, and—OC(O)R²⁰; and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —OR²⁰, —SR²⁰,—C(O)N(R²⁰)₂, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —OC(O)R²⁰, C₃₋₁₂carbocycle, and 3- to 12-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R²³ and R¹¹ taken together form an optionallysubstituted 5- to 6-membered heterocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), R¹¹ and R¹² taken together form an optionallysubstituted C₃₋₆ carbocycle.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), X² is C(O).

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), L³ is a cleavable linker. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), L³ is cleavable by a lysosomal enzyme.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), L³ is represented by the formula:

wherein:

L⁴ represents the C-terminus of the peptide and L⁵ is selected from abond, alkylene and heteroalkylene, wherein L⁵ is optionally substitutedwith one or more groups independently selected from R³⁰, and RX is areactive moiety; and

R³⁰ is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; and C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,and C₂-C₁₀alkynyl, each of which is independently optionally substitutedat each occurrence with one or more substituents selected from halogen,—OH, —CN, —O-alkyl, —SH, ═O, ═S, —NH₂, and —NO₂.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), RX comprises a leaving group. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), RX is a maleimide or an alpha-halo carbonyl. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), the peptide of L³ comprises Val-Cit or Val-Ala.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), L³ is represented by the formula:

wherein:

RX comprises a reactive moiety; and

n is 0-9.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), RX comprises a leaving group. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), RX is a maleimide or an alpha-halo carbonyl. In certainembodiments, for a compound or salt of any one of Formulas (IIA), (IIB),or (IIC), L³ is further covalently bound to an antibody or antigenbinding fragment thereof to form a conjugate.

In certain embodiments, the disclosure provides a conjugate representedby the formula:

wherein:

Antibody is an anti-Nectin-4 antibody or an antigen-binding fragmentthereof disclosed herein;

n is 1 to 20;

D is a compound or salt of any one of a Category B compound of Formulas(IA), (IB), or (IC); and L³ is a linker moiety; or

D-L³ is a compound or salt of any one of a Category B compound ofFormulas (IIA), (IIB), or (IIC).

In certain embodiments, for a conjugate of a compound or salt of any oneof Formulas (IA), (IB), (IC), (IIA), (IIB), and (IIC), n is selectedfrom 1 to 8. In certain embodiments, for a conjugate of a compound orsalt of any one of Formulas (IA), (IB), (IC), (IIA), (IIB), and (IIC), nis selected from 2 to 5. In certain embodiments, for a conjugate of acompound or salt of any one of Formulas (IA), (IB), (IC), (IIA), (IIB),and (IIC), n is 2.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), and (IIC), -L³ is represented by the formula:

wherein:

L⁴ represents the C-terminus of the peptide and L⁵ is selected from abond, alkylene and heteroalkylene, wherein L⁵ is optionally substitutedwith one or more groups independently selected from R³⁰;

RX* is a bond, a succinimide moiety, or a hydrolyzed succinimide moietybound to a residue of an antibody or antigen binding fragment thereof,wherein

on RX* represents the point of attachment to the residue of the antibodyor antigen binding fragment thereof; and

R³⁰ is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; and C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,and C₂-C₁₀ alkynyl, each of which is independently optionallysubstituted at each occurrence with one or more substituents selectedfrom halogen, —OH, —CN, —O-alkyl, —SH, ═O, ═S, —NH₂, and —NO₂.

In certain embodiments, for a compound or salt of any one of Formulas(IIA), (IIB), or (IIC), RX* is a succinamide moiety, hydrolyzedsuccinamide moiety or a mixture thereof and is bound to a cysteineresidue of an antibody.

In certain embodiments for a compound of Formulas (IIA), (IIB) and(IIC), -L³ is represented by the formula:

wherein:

RX* is a bond, a succinimide moiety, or a hydrolyzed succinimide moietybound to a residue of an antibody, wherein

a on RX* represents the point of attachment to the residue of theantibody; and

n is 0-9.

Examples of TLR7 agonist compounds according to Category B are providedin Table 3 and their stereoisomers. It is understood that salts of thecompounds provided in Table 3 are also envisioned by Table 3.

TABLE 3 Compounds 3.1-3.14 Compound Structure 3.1 

benzyl (1-((2-((1-(4-amino-2- (ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2- methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2-yl)carbamate 3.2 

2-amino-N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-methylpropanamide 3.3 

benzyl (S)-(1-((2-((1-(4-(2-((tert- butoxycarbonyl)amino)propanamido)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1- oxopropan-2-yl)carbamate3.4 

tert-butyl (2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2- methylpropan-2-yl)oxy)ethyl)carbamate3.5 

tert-butyl (2-((1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)carbamate 3.6 

benzyl (S)-(1-((2-((1-(4-(2-aminopropanamido)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2- yl)carbamate3.7 

tert-butyl2-(((S)-1-((2-(ethoxymethyl)-1-(5,5,11,11-tetramethyl-3,6-dioxo-1-phenyl-2,10-dioxa-4,7-diazadodecan-12-yl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-1-carboxylate 3.8 

(9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((1-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)-2- methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)carbamate 3.9 

3.10

3.11

3.12

3.13

3.14

In some aspects, the present disclosure provides a conjugate representedby the following structure:

or a pharmaceutically acceptable salt thereof, wherein Ab comprises ananti-Nectin-4 antibody or an antigen-binding fragment thereof disclosedherein, D is a compound or salt of a Category B compound of Formula(IID):

wherein R⁴ is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, or heteroarylalkyl group comprising from 1 to 8carbons, each J is hydrogen, each U is N, each t is 2, Q is not present,the dashed line represents a point of attachment of the adjuvant to G₁,and G₁ is a bond; subscript a is an integer from 1 to 40; and subscriptr is an integer from 1 to 10.

In certain embodiments, D has the following structure:

In further embodiments, the conjugate has the following structure:

In any of the aforementioned embodiments having a conjugate structureof:

wherein D is a compound or salt of a Category B compound of Formula(IID):

wherein R⁴ is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, or heteroarylalkyl group comprising from 1 to 8carbons, each J is hydrogen, each U is N, each t is 2, Q is not present,the dashed line represents a point of attachment of the adjuvant to G₁,and G₁ is a bond; subscript a is an integer from 1 to 40; and subscriptt r is an integer from 1 to 10; or

the antibody of the conjugate comprises a heavy chain variable region(VH) and a light chain variable region (VL),

(1) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence ofSEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:3; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence selected from any one of SEQ ID NOS:4-6, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:7, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:8;

(2) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:33, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:34, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:35; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:36, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:37, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:38;

(3) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:39, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:40, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:41; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:42, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:43, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:44;

(4) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:45, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:46, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:47; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:48, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:49, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:50;

(5) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:51, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:52, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:53; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:54, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:55, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:56;

(6) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:57, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:58, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:59; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:60, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:61, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:62;

(7) wherein VH comprises the amino acid sequence of SEQ ID NO: 10, andVL comprises the amino acid sequence selected from any one of SEQ IDNOS: 12-17; or

(8) wherein VH comprises the amino acid of SEQ ID NO:24, and VLcomprises the amino acid sequence selected from any one of SEQ IDNOS:26-31.

In another aspect, the present disclosure provides a conjugaterepresented by the following structure:

or a pharmaceutically acceptable salt thereof, wherein Ab comprises ananti-Nectin-4 antibody or an antigen-binding fragment thereof disclosedherein; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety; R¹is selected from H and C₁₋₄ alkyl; or Z, R¹, and the nitrogen atom towhich they are attached form a linking moiety comprising a 5- to8-membered heterocycle; each Y is independently CHR², wherein R² isselected from H, OH, and NH₂, R³ is selected from C₁₋₆ alkyl and 2- to6-membered heteroalkyl, each of which is optionally substituted with oneor more members selected from the group consisting of halo, hydroxy,amino, oxo (═O), alkylamino, amido, acyl, nitro, cyano, and alkoxy; X isselected from O and CH₂; subscript n is an integer from 1 to 12; andsubscript r is an integer from 1 to 10.

In certain embodiments, the conjugate is represented by the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein Ab comprises ananti-Nectin-4 antibody or an antigen-binding fragment thereof disclosedherein; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety; R¹is selected from H and C₁₋₄ alkyl; or Z, R¹, and the nitrogen atom towhich they are attached form a linking moiety comprising a 5- to8-membered heterocycle; each Y is independently CHR², wherein R² isselected from H, OH, and NH₂; X is selected from O and CH₂; subscript nis an integer from 1 to 12; and W is selected from the group consistingof O and CH₂.

In further embodiments, the conjugate is represented by the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein Ab comprises ananti-Nectin-4 antibody or antigen-binding fragment thereof disclosedherein; subscript r is an integer from 1 to 10; A is an unmodified aminoacid sidechain in the antibody or a modified amino acid sidechain in theantibody; Z is a linking moiety; and R¹ is selected from H and C₁₋₄alkyl; or Z, R¹, and the nitrogen atom to which they are attached form alinking moiety comprising a 5- to 8-membered heterocycle; and R² isselected from H, OH, and NH₂.

In yet further embodiments, the conjugate is represented by thefollowing structure:

or a pharmaceutically acceptable salt thereof, wherein Ab comprises ananti-Nectin-4 antibody or antigen-binding fragment thereof disclosedherein; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; R² is selected from H,OH, and NH₂; and subscript r is an integer from 1 to 10.

In any of the aforementioned embodiments having a conjugate structureof:

the antibody of the conjugate comprises a heavy chain variable region(VH) and a light chain variable region (VL),

(1) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence ofSEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:3; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence selected from any one of SEQ ID NOS:4-6, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:7, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:8;

(2) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:33, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:34, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:35; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:36, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:37, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:38;

(3) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:39, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:40, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:41; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:42, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:43, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:44;

(4) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:45, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:46, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:47; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:48, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:49, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:50;

(5) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:51, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:52, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:53; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:54, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:55, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:56;

(6) wherein the VH comprises a CDR1 (VH-CDR1) comprising the amino acidsequence of SEQ ID NO:57, a VH-CDR2 comprising the amino acid sequenceof SEQ ID NO:58, a VH-CDR3 comprising the amino acid sequence of SEQ IDNO:59; and the VL comprises a CDR1 (VL-CDR1) comprising the amino acidsequence of SEQ ID NO:60, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:61, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:62;

(7) wherein VH comprises the amino acid sequence of SEQ ID NO: 10, andVL comprises the amino acid sequence selected from any one of SEQ IDNOS:12-17; or

(8) wherein VH comprises the amino acid of SEQ ID NO:24, and VLcomprises the amino acid sequence selected from any one of SEQ IDNOS:26-31.

Compounds of Category C, TLR8 Agonists

In some aspects, the myeloid cell agonist is a benzazepine compound(Bza). In some aspects, the present disclosure provides a conjugaterepresented by Formula I:

wherein: A is an anti-Nectin-4 antibody or an antigen-binding fragmentthereof, L is a linker; D_(x) is an immune-stimulatory compound; n isselected from 1 to 20; and z is selected from 1 to 20.

In certain embodiments of the conjugates of the disclosure, D_(x) isselected from a compound or salt of a compound of the disclosure,including, but not limited to Category C (e.g., Formulas (Ia), (Ib),(Ic), (Id), (Ie), (If), (Ig) and (Ih)).

In certain embodiments, L is represented by the formula:

wherein

L⁴ represents the C-terminal of the peptide and

L⁵ is selected from a bond, alkylene and heteroalkylene,

-   -   wherein L⁵ is optionally substituted with one or more groups        independently selected from R³²;

RX* comprises a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of an antibody or antigen binding fragmentthereof,

-   -   wherein

-   -    on RX* represents the point of attachment to the residue of the        antibody or antigen binding fragment thereof; and,

R³² is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; and C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OH, —CN, —O-alkyl,—SH, ═O, ═S, —NH₂, —NO₂. In some embodiments, the peptide of L comprisesVal-Cit or Val-Ala.

In certain embodiments of the Category C compounds of the disclosure,D_(x) comprises an aminobenzazepine moiety having the formula:

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, C₁-C₁₂ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₂ carbocyclyl,C₆-C₂₀ aryl, C₂-C₉ heterocyclyl, and C₁-C₂₀ heteroaryl, where alkyl,alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl areindependently and optionally substituted with one or more groupsselected from: —(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-N(R⁵)₂;—(C₃-C₁₂ carbocyclyl); —(C₃-C₁₂ carbocyclyl)-*; —(C₃-C₁₂carbocyclyl)-(C₁-C₁₂ alkyldiyl)-NR⁵—*; —(C₃-C₁₂ carbocyclyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —(C₃-C₁₂ carbocyclyl)-NR⁵—C(═NR⁵)NR⁵—*; —(C₆-C₂₀aryl); —(C₆-C₂₀ aryl)-*; —(C₆-C₂₀ aryldiyl)-N(R⁵)—*; —(C₆-C₂₀aryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₆-C₂₀ aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —(C₆-C₂₀aryldiyl)-(C₁-C₁₂alkyldiyl)-NR⁵—C(═NR^(5a))N(R⁵)—*; —(C₂-C₂₀ heterocyclyl); —(C₂-C₂₀heterocyclyl)-*; —(C₂-C₉ heterocyclyl)-(C₁-C₁₂ alkyldiyl)-NR₅—*; —(C₂-C₉heterocyclyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —(C₂-C₉heterocyclyl)-NR⁵—C(═NR^(5a))NR⁵—*; —(C₁-C₂₀ heteroaryl); —(C₁-C₂₀heteroaryl)-*; —(C₁-C₂₀ heteroaryl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₁-C₂₀heteroaryl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —(C₁-C₂₀heteroaryl)-NR⁵—C(═NR^(5a))N(R⁵)—*; —C(═O)—*; —C(═O)—(C₂-C₂₀heterocyclyldiyl)-*; —C(═O)N(R⁵)₂; —C(═O)N(R⁵)—*; —C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)R⁵; —C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)N(R⁵)₂; —C(═O)NR⁵—(C₁-C₁₂ alkyldiyl)-N(R⁵)CO₂R⁵;—C(═O)NR⁵—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═NR5a)N(R⁵)₂; —C(═O)NR⁵—(C₁-C₁₂alkyldiyl)-NR⁵C(═NR^(5a))R⁵; —C(═O)NR⁵—(C₁-C₅ alkyldiyl)-NR⁵ (C₂-C₅heteroaryl); —C(═O)NR⁵—(C₁-C₂₀ heteroaryldiyl)-N(R⁵)—*;—C(═O)NR⁵—(C₁-C₂₀ heteroaryldiyl)-*; —C(═O)NR⁵—(C₁-C₂Oheteroaryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —C(═O)NR⁵—(C₁-C₂₀heteroaryldiyl)-(C₂-C₂₀ heterocyclyldiyl)-C(═O)NR⁵—(C₁-C₁₂alkyldiyl)-NR⁵*; —N(R⁵)₂; —N(R⁵)—*; —N(R⁵)C(═O) R⁵; —N(R⁵)C(═O)—*;—N(R⁵)C(═O)N(R⁵)₂; —N(R⁵)C(═O)N(R⁵)—*; —N(R⁵)CO₂R⁵;—NR⁵C(═NR^(5a))N(R⁵)₂; —NR⁵C(═NR^(5a))N(R⁵)—*; —NR⁵C(═NR^(5a)) R⁵;—N(R⁵)—(C₂-C₅ heteroaryl); —O—(C₁-C₁₂ alkyl); —O—(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —O—(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —S(═O)₂—(C₂-C₂₀heterocyclyldiyl)-*; —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-NR⁵*; and —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-OH; or R² and R³ together form a 5- or 6-memberedheterocyclyl ring;

X¹, X², X³, and X⁴ are independently selected from the group consistingof a bond, C(═O), C(═O)N(R⁵), O, N(R⁵), S, S(O)₂, and S(O)₂N(R⁵); R⁵ isselected from the group consisting of H, C₆-C₂₀ aryl, C₆-C₂₀ aryldiyl,C₁-C₁₂ alkyl, and C₁-C₁₂ alkyldiyl, or two Rs groups together form a 5-or 6-membered heterocyclyl ring;

R^(5a) is selected from the group consisting of C₆-C₂₀ aryl and C₁-C₂₀heteroaryl;

where the asterisk* indicates the attachment site of L, and where one ofR¹, R², R³ and R⁴ is attached to L;

L is the linker selected from the group consisting of: —C(═O)-(PEG)-;—C(═O)-(PEG)-C(═O)—; —C(═O)-(PEG)-O—; —C(═O)-(PEG)-C(═O)-(PEP)-;—C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂ alkyldiyl)-;—C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅monoheterocyclyldiyl)-; —C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-(MCgluc)-; —C(═O)-(PEG)-C(═O)-(MCgluc)-;—C(═O)-(PEG)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-;—C(═O)-(PEG)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅monoheterocyclyldiyl)-; —C(═O)-(PEG)-N(R⁵)—;—C(═O)-(PEG)-N(R⁵)—(PEG)-C(═O)-(PEP)-;—C(═O)-(PEG)-N+(R⁵)₂-(PEG)-C(═O)-(PEP)-;—C(═O)-(PEG)-C(═O)—N(R⁵)CH(AA₁)C(═O)-(PEG)-C(═O)-(PEP)-;—C(═O)-(PEG)-C(═O)—N(R⁵)CH(AAI)C(═O)—N(R⁵)—(C₁-C₁₂ alkyldiyl)-;—C(═O)-(PEG)-SS—(C₁-C₁₂ alkyldiyl)-OC(═O)—; —C(═O)-(PEG)-SS—(C₁-C₁₂alkyldiyl)-C(═O)—; —C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-;—C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-;—C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)—C(═O);—C(═O)—(C₁-C₁₂alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅monoheterocyclyldiyl)-; —C(═O)—CH₂CH₂OCH₂CH₂—(C₁-C₂₀heteroaryldiyl)-CH₂O—(PEG)-C(═O)(MCgluc)-; —C(═O)—CH₂CH₂OCH₂CH₂—(C₁-C₂₀heteroaryldiyl)-CH₂O—(PEG)-C(═O)(MCgluc)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅ monoheterocyclyldiyl)-; and-(succinimidyl)-(CH₂)_(m)—C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅ monoheterocyclyldiyl)-;

PEG has the formula: —(CH₂CH₂O)_(n)—(CH₂)_(m)—; m is an integer from 1to 5, and n is an integer from 2 to 50;

PEP has the formula:

where AA₁ and AA₂ are independently selected from an amino acid sidechain, or AA₁ or AA₂ and an adjacent nitrogen atom form a 5-memberedring praline amino acid, and the wavy line indicates a point ofattachment;

R⁶ is selected from the group consisting of C₆-C₂₀ aryldiyl and C₁-C₂₀heteroaryldiyl, substituted with —CH₂O—C(═O)— and optionally with:

and

MCgluc is selected from the groups:

where q is 1 to 8, and AA is an amino acid side chain; and

alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl,aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl,heteroaryl, and heteroaryldiyl are independently and optionallysubstituted with one or more groups independently selected from F, Cl,Br, I, —CN, —CH₃, —CH₂CH₃, —CH═CH₂, —C═CH, —C═CCH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH, —C(CH₃)₂OH,—CH(OH)CH(CH₃)₂, —C(CH₃)₂CH₂OH, —CH₂CH₂SO₂CH₃, —CH₂OP(O)(OH)₂, —CH₂F,—CHF₂, —CF₃, —CH₂CF₃, —CH₂CHF₂, —CH(CH₃)CN, —C(CH₃)₂CN, —CH₂CN, —CH₂NH₂,—CH₂NHSO₂CH₃, —CH₂NHCH₃, —CH₂N(CH₃)₂, —CO₂H, —COCH₃, —CO₂CH₃,—CO₂C(CH₃)₃, —COCH(OH)CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂,—NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —N(CH₃)COCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, —NHC(═NH)H, —NHC(═NH)CH₃,—NHC(═NH)NH₂, —NHC(═O)NH₂, —NO₂, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OCH₃,—OCH₂CH₂OH, —OCH₂CH₂N(CH₃)₂, —O(CH₂CH₂O)n-(CH₂)mCO₂H, —O(CH2CH2O)_(n)H,—OP(O)(OH)₂, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, and —S(O)₃H.

In certain embodiments, the PEP is selected from the groups:

wherein n is 1 or more and AA is an amino acid side chain. In certainembodiments of Formula I, each of AA₁ and AA₂ are independently selectedfrom a side chain of a naturally-occurring amino acid. In certainembodiments of Formula I, each of AA₁ and AA₂ are independently selectedfrom H, —CH₃, —CH(CH₃)₂, —CH₂(C₆H₅), —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂NHC(NH)NH₂, —CH₂CH(CH₃)₂, —CH₂SO₃H, and —CH₂CH₂CH₂NHC(O)NH₂.In certain embodiments of Formula I, AA₁ is —CH(CH₃)₂ and AA₂ is—CH₂CH₂CH₂NHC(O)NH₂. In certain embodiments of Formula I, each of AA₁and AA₂ are independently selected from GlcNAc, aspartic acid, —CH₂SO₃Hand —CH₂OPO₃H.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, L-D_(x) is selected from Formulas Ia-Id:

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, L is —C(═O)-(PEG)- or —C(═O)-(PEG)-C(═O)—. Incertain embodiments of the formulas of the disclosure, including FormulaI of Category C, L-D_(x) is selected from Formulas Ie and If:

wherein R^(5a) of Formula If is phenyl, optionally substituted with oneor more groups selected from F, Cl, Br, I, —CN, and —NO₂. In certainembodiments of the formulas of the disclosure, including Formula I ofCategory C, L-D_(x) is selected from Formulas Ig and Ih:

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, L is —C(═O)-(PEG)-C(═O)-(PEP)-.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, R² and R³ are each C₁-C₅ alkyl.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, R² and R³ are each —CH₂CH₂CH₃.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, X² and X³ are each a bond, and R² or R³ is—O—(C₁-C₁₂ alkyl).

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, X² and X³ are each a bond, and R² or R³ is—OCH₂CH₃.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, one of R1 and R4 is selected from: —(C₁-C₁₂alkyldiyl)-N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═NR⁵)N(R⁵)—*; —(C₆-C₂₀aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₆-C₂₀aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*;—(C₆-C₂₀aryldiyl)-C(═O)—*; —(C₆-C₂₀ aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)—*; —(C₆-C₂₀ aryldiyl)-C(═O)—(C₂-C₂₀heterocyclyldiyl)-*; —C(═O)NR⁵—(C₁-C₂O heteroaryldiyl)-*; and—C(═O)NR⁵—(C₁-C₂O heteroaryldiyl)-(C₂-C₂₀heterocyclyldiyl)-C(═O)NR⁵—(C₁-C₁₂ alkyldiyl)-NR⁵—*.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, one of R² and R³ is selected from: —(C₁-C₁₂alkyldiyl)-N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-O—(C₁-C₁₂ alkyldiyl)-N(R⁵)—*;—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═NR⁵)—N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-(C₆-C₂₀aryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-(C₆-C₂₀aryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—C(═NR⁵)N(R⁵)—*; —(C₂-C₆alkynyldiyl)-N(R⁵)—*; and —(C₂-C₆ alkynyldiyl)-N(R⁵)C(═NR⁵)N(R⁵)—*; X²and X³ are a bond, and where the asterisk* indicates the attachment siteof L.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, one of R¹ and R⁴ is selected from —(C₆-C₂₀aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂ and—(C₆-C₂₀ aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-OH.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, C₆-C₂₀ aryldiyl is phenyldiyl and C₂-C₂₀heterocyclyldiyl is azetidindiyl.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, one of R¹ and R⁴ is selected from the formulas:

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, one of R¹ and R⁴ is —C(═O)NR⁵—(C₁-C₂₀heteroaryldiyl)-(C₂-C₂₀ heterocyclyldiyl)-C(═O)NR⁵—(C₁-C₁₂alkyldiyl)-NR⁵-L.

In certain embodiments of the formulas of the disclosure, includingFormula I of Category C, C₁-C₂₀ heteroaryldiyl is pyridindiyl and C₂-C₂₀heterocyclyldiyl is piperidiyl.

In some aspects, the disclosure provides a conjugate comprising abenzazepine according to Formula (II):

wherein

Z is selected from H, —O(C₁-C₈ alkyl), and N(X²R²)(X³R³); R¹, R², R³,and R⁴ are independently selected from the group consisting of H, C₁-C₁₂alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₂ carbocyclyl, C₆-C₂₀ aryl,C₂-C₉ heterocyclyl, and C₁-C₂₀ heteroaryl, where alkyl, alkenyl,alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl areindependently and optionally substituted with one or more groupsselected from: —(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₁-C₁₂ alkyldiyl)-N(R⁵)₂;—(C₃-C₁₂ carbocyclyl); —(C₃-C₁₂ carbocyclyl)-*; —(C₃-C₁₂carbocyclyl)-(C₁-C₁₂ alkyldiyl)-NR⁵—*; —(C₃-C₁₂ carbocyclyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —(C₃-C₁₂ carbocyclyl)-NR⁵—C(═NR⁵)NR⁵—*; —(C₆-C₂₀aryl); —(C₆-C₂₀ aryl)-*; —(C₆-C₂₀ aryldiyl)-N(R⁵)—*; —(C₆-C₂₀aryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₆-C₂₀ aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —(C₆-C₂₀aryldiyl)-(C₁-C₁₂alkyldiyl)-NR⁵—C(═NR^(5a))N(R⁵)—*; —(C₂-C₂₀ heterocyclyl); —(C₂-C₂₀heterocyclyl)-*; —(C₂-C₉ heterocyclyl)-(C₁-C₁₂ alkyldiyl)-NR⁵—*; —(C₂-C₉heterocyclyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —(C₂-C₉heterocyclyl)-NR⁵—C(═NR^(5a))NR⁵—*; —(C₁-C₂₀ heteroaryl); —(C₁-C₂₀heteroaryl)-*; —(C₁-C₂₀ heteroaryl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —(C₁-C₂₀heteroaryl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —(C₁-C₂₀heteroaryl)-NR⁵—C(═NR^(5a))N(R⁵)—*; —C(═O)—*; —C(═O)—(C₂-C₂₀heterocyclyldiyl)-*; —C(═O)N(R⁵)₂; —C(═O)N(R⁵)—*; —C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)R⁵; —C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)N(R⁵)₂; —C(═O)NR⁵—(C₁-C₁₂ alkyldiyl)-N(R⁵)CO₂R⁵;—C(═O)NR⁵—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═NR^(5a))N(R⁵)₂; —C(═O)NR⁵—(C₁-C₁₂alkyldiyl)-NR⁵C(═NR^(5a)a)R⁵; —C(═O)NR⁵—(C₁-C₈ alkyldiyl)-NR⁵ (C₂-C₈heteroaryl); —C(═O)NR⁵—(C₁-C₂O heteroaryldiyl)-N(R⁵)—*;—C(═O)NR⁵—(C₁-C₂₀ heteroaryldiyl)-*; —C(═O)NR⁵—(C₁-C₂Oheteroaryldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂; —C(═O)NR⁵—(C₁-C₂₀heteroaryldiyl)-(C₂-C₂₀ heterocyclyldiyl)-C(═O)NR⁵—(C₁-C₁₂alkydiyl)-NR⁵; —N(R⁵)₂; —N(R⁵)—*; —N(R⁵)C(═O)R⁵; —N(R⁵)C(═O)—*;—N(R⁵)C(═O)N(R⁵)₂; —N(R⁵)C(═O)N(R⁵)—*; —N(R⁵)CO₂R⁵;—NR⁵C(═NR^(5a))N(R⁵)₂; —NR⁵C(═NR^(5a))N(R⁵)—*; —NR⁵C(═NR^(5a))R⁵;—N(R⁵)—(C₂-C₈ heteroaryl); —O—(C₁-C₁₂ alkyl); —O—(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —O—(C₁-C₁₂ alkyldiyl)-N(R⁵)—*; —S(═O)₂—(C₂-C₂₀heterocyclyldiyl)-*; —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁵)₂; —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-NR⁵—*; and —S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-OH; or R² and R³ together form a 5- or 6-memberedheterocyclyl ring;

X¹, X², X³, and X⁴ are independently selected from the group consistingof a bond, C(═O), C(═O)N(R⁵), O, N(R⁵), S, S(O)₂, and S(O)₂N(R⁵);

R⁵ is selected from the group consisting of H, C₆-C₂₀ aryl, C₆-C₂₀aryldiyl, C₁-C₁₂ alkyl, and C₁-C₁₂ alkyldiyl, or two R⁵ groups togetherform a 5- or 6-membered heterocyclyl ring;

R^(5a) is selected from the group consisting of C₆-C₂₀ aryl and C₁-C₂₀heteroaryl;

where the asterisk* indicates the attachment site of L, and where one ofR¹, R², R³ and R⁴ is attached to L;

L is the linker selected from the group consisting of: Q-C(═O)-(PEG)-;Q-C(═O)-(PEG)-C(═O)—; Q-C(═O)-(PEG)-O—; Q-C(═O)-(PEG)-C(═O)-(PEP)-;Q-C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂ alkyldiyl)-;Q-C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)—(C₂-C₈monoheterocyclyldiyl)-; Q-C(═O)-(PEG)-C(═O)N(R⁵)—(C₁-C₁₂alkyldiyl)-(MCgluc)-; Q-C(═O)-(PEG)-C(═O)-(MCgluc)-;Q-C(═O)-(PEG)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-;Q-C(═O)-(PEG)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)—(C₂-C₈monoheterocyclyldiyl)-; Q-C(═O)-(PEG)-N(R⁵)—;Q-C(═O)-(PEG)-N(R⁵)—(PEG)-C(═O)-(PEP)-;Q-C(═O)-(PEG)-N⁺(R⁵)₂-(PEG)-C(═O)-(PEP)-;Q-C(═O)-(PEG)-C(═O)—N(R⁵)CH(AA₁)C(═O)-(PEG)-C(═O)-(PEP)-;Q-C(═O)-(PEG)-C(═O)—N(R⁵)CH(AAI)C(═O)—N(R⁵)—(C₁-C₁₂ alkyldiyl)-;Q-C(═O)-(PEG)-SS—(C₁-C₁₂ alkyldiyl)-OC(═O)—; Q-C(═O)-(PEG)-SS—(C₁-C₁₂alkyldiyl)-C(═O)—; Q-C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-;Q-C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-;Q-C(═O)—(C₁-C₁₂ alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)—C(═O); Q-C(═O)—(C₁-C₁₂alkyldiyl)-C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)(C₂-C₅monoheterocyclyldiyl)-; Q-C(═O)—CH₂CH₂OCH₂CH₂—(C₁-C₂₀heteroaryldiyl)-CH₂O—(PEG)-C(═O)(MCgluc)-; Q-C(═O)—CH₂CH₂OCH₂CH₂—(C₁-C₂Oheteroaryldiyl)-CH₂O—(PEG)-C(═O)(MCgluc)-N(R⁵)—(C₁-C₁₂alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅ monoheterocyclyldiyl)-; andQ-(CH₂)_(m)—C(═O)-(PEP)-N(R⁵)—(C₁-C₁₂ alkyldiyl)-N(R⁵)C(═O)—(C₂-C₅monoheterocyclyldiyl)-;

where PEG has the formula: —(CH₂CH₂O)_(n)—(CH₂)_(m)—; m is an integerfrom 1 to 5, and

n is an integer from 2 to 50;

PEP has the formula:

where AA₁ and AA₂ are independently selected from an amino acid sidechain, or

AA₁ or AA₂ and an adjacent nitrogen atom form a 5-membered ring pralineamino acid, and the wavy line indicates a point of attachment and;

R⁶ is selected from the group consisting of C₆-C₂₀ aryldiyl and C₁-C₂₀heteroaryldiyl, substituted with —CH₂O—C(═O)— and optionally with:

and

MCgluc is selected from the groups:

where q is 1 to 8, and AA is an amino acid side chain; and

Q is selected from the group consisting of N-hydroxysuccinimidyl,N-hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with oneor more groups independently selected from F, Cl, NO₂, and SO₃ ⁻;

where alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl,aryl, aryldiyl carbocyclyl, carbocyclyldiyl, heterocyclyl,heterocyclyldiyl, heteroaryl, and heteroaryldiyl are optionallysubstituted with one or more groups independently selected from F, Cl,Br, I, —CN, —CH₃, —CH₂CH₃, —CH═CH₂, —C═CH, —C═CCH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH, —C(CH₃)₂OH,—CH(OH)CH(CH₃)₂, —C(CH₃)₂CH₂OH, —CH₂CH₂SO₂CH₃, —CH₂OP(O)(OH)₂, —CH₂F,—CHF₂, —CF₃, —CH2CF3, —CH₂CHF₂, —CH(CH₃)CN, —C(CH₃)₂CN, —CH₂CN, —CH₂NH₂,—CH₂NHSO₂CH₃, —CH₂NHCH₃, —CH₂N(CH₃)₂, —CO₂H, —COCH₃, —CO₂CH₃,—CO₂C(CH₃)₃, —COCH(OH)CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂,—NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —N(CH₃)COCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, —NHC(═NH)H, —NHC(═NH)CH₃,—NHC(═NH)NH₂, —NHC(═O)NH₂, —NO₂, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OCH₃,—OCH₂CH₂OH, —OCH₂CH₂N(CH₃)₂, —O(CH₂CH₂O)_(n)—(CH₂)_(m)CO₂H,—O(CH₂CH₂O)_(n)H, —OP(O)(OH)₂, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, and—S(O)₃H.

In certain embodiments of the formulas of the disclosure, includingFormula (II) of Category C, PEP is selected from the groups:

wherein n is 1 or more and AA is an amino acid side chain.

In certain embodiments of the formulas of the disclosure, includingFormula (II) of Category C, each AA₁ and AA₂ are independently selectedfrom a side chain of a naturally-occurring amino acid.

In certain embodiments of the formulas of the disclosure, includingFormula (II) of Category C, AA₁ and AA₂ are independently selected fromH, —CH₃, —CH(CH₃)₂, —CH₂(C₆H₅), —CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂,—CH₂CH(CH₃)₂, —CH₂SO₃H, and —CH₂CH₂CH₂NHC(O)NH₂.

In certain embodiments of the formulas of the disclosure, includingFormula (II) of Category C, each AA₁ is —CH(CH₃)₂, and AA₂ is—CH₂CH₂CH₂NHC(O)NH₂.

In certain embodiments of the formulas of the disclosure, includingFormula (II) of Category C, each AA₁ and AA₂ are independently selectedfrom GlcNAc aspartic acid, —CH₂SO₃H, and —CH₂OPO₃H.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, the aminobenzazepine-linker compound ofFormula (II) is selected from Formulas IIa-IId:

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, the aminobenzazepine-linker compound ofFormula (II) is selected from Formulas IIe and IIf:

wherein R^(5a) of formula Ilf is phenyl, optionally substituted with oneor more groups selected from F, Cl, Br, I, —CN, and —NO₂.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, L is Q-C(═O)-(PEG)- or Q-C(═O)-(PEG)-C(═O)—.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, the aminobenzazepine-linker compound ofFormula II is selected from Formulas IIg and IIh:

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, L is —C(═O)-(PEG)-C(═O)-(PEP)-.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, R² and R³ are each C₁-C₈ alkyl.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, R² and R³ are each —CH₂CH₂CH₃.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, X² and X³ are each a bond, and R² and R³ is—O—(C₁-C₁₂ alkyl).

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, X² and X³ are each a bond, and R² and R³ is—OCH₂CH₃.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, one of R¹ and R⁴ is selected from —(C₆-C₂₀aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂ alkyldiyl)-N(R⁵)₂ and—(C₆-C₂₀ aryldiyl)-S(═O)₂—(C₂-C₂₀ heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-OH.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, C₆-C₂₀ aryldiyl is phenyldiyl and C₂-C₂₀heterocyclyldiyl is azetidindiyl.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, the aminobenzazepine-linker compound ofFormula II is selected from Formulas:

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, one of R¹ and R⁴ is —C(═O)NR⁵—(C₁-C₂₀heteroaryldiyl)-(C₂-C₂₀ heterocyclyldiyl)-C(═O)NR⁵—(C₁-C₁₂alkyldiyl)-NR⁵-L.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, C₁-C₂₀ heteroaryldiyl is pyridindiyl andC₂-C₂₀ heterocyclyldiyl is piperidiyl.

In certain embodiments of the formulas of the disclosure, includingFormula II of Category C, Q is selected from:

In some aspects, the disclosure provides a conjugate comprising abenzazepine according to Formula III:

a pharmaceutically acceptable salt thereof, or a quaternary ammoniumsalt thereof, wherein

R¹, R², R³, and R⁴ are independently Y or Z, wherein one of R¹, R², R³,and R⁴ is Y, having the formula:

independently is hydrogen or selected from the formulas:

U is optionally present and is CH₂, C(═O), CH₂C(═O), or C(═O)CH₂,

A is optionally present and is NR¹⁰ or selected from the formulas:

R¹⁰ and W independently are hydrogen, Ar¹, or of formula:

V is optionally present and is of formula:

J¹ and J² independently are CH or N,

m¹, m², and m³ independently are an integer from 0 to 25, except that atleast one of m¹, m², and m³ is a non-zero integer,

n¹, n², n³, n⁴, n⁵, and n⁶ independently are an integer from 0 to 10,

t₁ and t₂ independently are an integer from 1 to 3,

G₁, G₂, G₃, and G₄ independently are CH₂, C(═O), CH₂C(═O), C(═O)CH₂, ora bond,

X¹, X², X³, and X⁴ are each optionally present and independently are O,NR⁷, CHR⁷, SO₂, S, or one or two cycloalkyldiyl, heterocycloalkyldiyl,aryldiyl, or heteroaryldiyl groups, and when more than onecycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groupis present, the more than one cycloalkyldiyl, heterocycloalkyldiyl,aryldiyl, or heteroaryldiyl groups are linked or fused, wherein linkedcycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groupsare linked through a bond or —CO—,

R⁹ is hydrogen, C₁-C₄ alkyl, or selected from the formulas:

R⁸ is independently hydrogen or C₁-C₄ alkyl,

Ar¹ and Ar² independently are an aryl or heteroaryl group, optionallysubstituted with one or more halogens (e.g., fluorine, chlorine,bromine, or iodine), nitriles, hydroxyls, C₁-C₄ alkyl groups, or acombination thereof,

L_(M) is a linking moiety that comprises a functional group selectedfrom an amide,

amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate,and thiourea,

r is an integer from 1 to 50,

“Ms” is a macromolecular support, and

each wavy line (˜) represents a point of attachment.

Nonlimiting examples of TLR8 agonist compounds of Category C areprovided in Table 1b.

TABLE 1b Compounds 1.70-1.74 Compound Structure 1.70

1.71

1.72

1.73

1.74

Linkers

The conjugates include a linker(s) that attaches an anti-Nectin-4antibody or antigen-binding fragment thereof to at least oneimmune-stimulatory compound, such as a myeloid cell agonist. A linkercan be, for example, a cleavable or a non-cleavable linker. A conjugatecan comprise multiple linkers. The linkers in a conjugate can be thesame linkers or different linkers.

As will be appreciated by skilled artisans, a linker connects animmune-stimulatory compound(s), such as a myeloid cell agonist, to theantibody or antigen-binding fragment thereof by forming a covalentlinkage to the compound at one location and a covalent linkage to theantibody or antigen-binding fragment thereof at another location. Thecovalent linkages can be formed by reaction between functional groups onthe linker and functional groups on the immune-stimulatory compound andon the antibody or antigen-binding fragment thereof. As used herein, theexpression “linker” can include (i) unattached forms of the linker thatcan include a functional group capable of covalently attaching thelinker to an immune-stimulatory compound and a functional group capableof covalently attached the linker to an antibody or antigen-bindingfragment thereof; (ii) partially attached forms of the linker that caninclude a functional group capable of covalently attaching the linker toan antibody or antigen-binding fragment thereof and that can becovalently attached to an immune-stimulatory compound, or vice versa;and (iii) fully attached forms of the linker that can be covalentlyattached to both an immune stimulatory compound and to an antibody orantigen-binding fragment thereof. In some specific embodiments, thefunctional groups on a linker and covalent linkages formed between thelinker and an antibody or antigen-binding fragment thereof can bespecifically illustrated as Rx and Rx′, respectively.

A linker can be short or long, and cleavable or non-cleavable. A linkercan contain segments that have different characteristics, such assegments of flexibility or segments of rigidity, segments ofhydrophilicity, and/or segments of hydrophobicity. A linker can bechemically stable to extracellular environments, for example, chemicallystable in the blood stream, and/or may include linkages that are notstable. A linker can include linkages that are designed to cleave and/orimmolate or otherwise breakdown specifically or non-specifically insidecells. A cleavable linker can be sensitive to enzymes at a specificsite, such as the lysosome or the extracellar space adjacent cancercells.

A cleavable linker can include a valine-citrulline peptide, avaline-alanine peptide, a phenylalanine-lysine or other peptide, such asa peptide that forms a protease recognition and cleavage site. Such apeptide-containing linker can contain a pentafluorophenyl group. Apeptide-containing linker can include a succimide or a maleimide group.A peptide-containing linker can include a para aminobenzoic acid (PABA)group. A peptide-containing linker can include an aminobenzyloxycarbonyl(PABC) group. A peptide-containing linker can include a PABA or PABCgroup and a pentafluorophenyl group. A peptide-containing linker caninclude a PABA or PABC group and a succinimide group. Apeptide-containing linker can include a PABA or PABC group and amaleimide group.

A non-cleavable linker is generally protease-insensitive and insensitiveto intracellular processes. A non-cleavable linker can include amaleimide group. A non-cleavable linker can include a succinimide group.A non-cleavable linker can be maleimido-alkyl-C(O)— linker. Anon-cleavable linker can be maleimidocaproyl linker. A maleimidocaproyllinker can be N-maleimidomethylcyclohexane-1-carboxylate. Amaleimidocaproyl linker can include a succinimide group. Amaleimidocaproyl linker can include pentafluorophenyl group.

A linker can be a combination of a maleimidocaproyl group and one ormore polyethylene glycol molecules. A linker can be a maleimide-PEG4linker. A linker can be a combination of a maleimidocaproyl linkercontaining a succinimide group and one or more polyethylene glycolmolecules. A linker can be a combination of a maleimidocaproyl linkercontaining a pentafluorophenyl group and one or more polyethylene glycolmolecules. A linker can contain a maleimide(s) linked to polyethyleneglycol molecules in which the polyethylene glycol can allow for morelinker flexibility or can be used lengthen the linker.

A linker can be a(maleimidocaproyl)-(valine-alanine)-(para-aminobenzyloxycarbonyl)linker. A linker can be a(maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl)linker. A linker can be a(maleimidocaproyl)-(phenylalanine-lysine)-(para-aminobenzyloxycarbonyl)linker.

A linker can also contain segments of alkylene, alkenylene, alkynylene,polyether, polyester, polyamide, polyamino acids, peptides,polypeptides, cleavable peptides, and/or aminobenzyl-carbamates. Alinker can contain a maleimide at one end and an N-hydroxysuccinimidylester at the other end. A linker can contain a lysine with an N-terminalamine acetylated, and a valine-citrulline, valine-alanine orphenylalanine-lysine cleavage site. A linker can be a link created by amicrobial transglutaminase, wherein the link can be created between anamine-containing moiety and a moiety engineered to contain glutamine asa result of the enzyme catalyzing a bond formation between the acylgroup of a glutamine side chain and the primary amine of a lysine chain.A linker can contain a reactive primary amine. A linker can be a SortaseA linker. A Sortase A linker can be created by a Sortase A enzyme fusingan LXPTG recognition motif (SEQ ID NO:32) to an N-terminal GGG motif toregenerate a native amide bond. The linker created can therefore link toa moiety attached to the LXPTG recognition motif (SEQ ID NO:32) with amoiety attached to the N-terminal GGG motif. A linker can be a linkcreated between an unnatural amino acid on one moiety reacting withoxime bond that was formed by modifying a ketone group with analkoxyamine on another moiety. A moiety can be part of a conjugate. Amoiety can be part of an antibody, such as an antibody. A moiety can bepart of an immune-stimulatory compound, such as a myeloid cell agonist.A moiety can be part of a binding domain. A linker can be unsubstitutedor substituted, for example, with a substituent. A substituent caninclude, for example, hydroxyl groups, amino groups, nitro groups, cyanogroups, azido groups, carboxyl groups, carboxaldehyde groups, iminegroups, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups,acyl groups, acyloxy groups, amide groups, and ester groups.

A linker can be polyvalent such that it covalently links more than oneimmune-stimulatory compound to a single site on the antibody orantigen-binding fragment thereof, or monovalent such that it covalentlylinks a single immune-stimulatory compound to a single site on theantibody or antigen-binding fragment thereof.

Exemplary polyvalent linkers that may be used to attach manyimmune-stimulatory compounds to an antibody or antigen-binding fragmentthereof of the conjugate are described. For example, Fleximer® linkertechnology has the potential to enable high-DAR conjugate with goodphysicochemical properties. As shown below, the Fleximer® linkertechnology is based on incorporating molecules into a solubilizingpoly-acetal backbone via a sequence of ester bonds. The methodologyrenders highly-loaded conjugates (DAR up to 20) whilst maintaining goodphysicochemical properties.

This methodology can be utilized with an immune-stimulatory compound asshown in the scheme below, where Drug' refers to the immune-stimulatorycompound.

To utilize the Fleximer® linker technology depicted in the scheme above,an aliphatic alcohol can be present or introduced into theimmune-stimulatory compound. The alcohol moiety is then attached to analanine moiety, which is then synthetically incorporated into theFleximer® linker. Liposomal processing of the conjugate in vitroreleases the parent alcohol-containing drug.

By way of example and not limitation, some cleavable and noncleavablelinkers that may be included in the conjugates described herein aredescribed below.

Cleavable linkers can be cleavable in vitro and in vivo. Cleavablelinkers can include chemically or enzymatically unstable or degradablelinkages. Cleavable linkers can rely on processes inside the cell toliberate an immune-stimulatory compound, such as reduction in thecytoplasm, exposure to acidic conditions in the lysosome, or cleavage byspecific proteases or other enzymes within the cell. Cleavable linkerscan incorporate one or more chemical bonds that are chemically orenzymatically cleavable while the remainder of the linker can benon-cleavable.

A linker can contain a chemically labile group such as hydrazone and/ordisulfide group. Linkers comprising chemically labile groups can exploitdifferential properties between the plasma and some cytoplasmiccompartments. The intracellular conditions that can facilitateimmune-stimulatory compound release for hydrazine-containing linkers canbe the acidic environment of endosomes and lysosomes, whiledisulfide-containing linkers can be reduced in the cytosol, which cancontain high thiol concentrations, e.g., glutathione. The plasmastability of a linker containing a chemically labile group can beincreased by introducing steric hindrance using substituents near thechemically labile group.

Acid-labile groups, such as hydrazones, can remain intact duringsystemic circulation in the blood's neutral pH environment (pH 7.3-7.5)and can undergo hydrolysis and can release an immune-stimulatorycompound once the conjugate is internalized into mildly acidic endosomal(pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell. ThispH dependent release mechanism can be associated with nonspecificrelease of the immune-stimulatory compound. To increase the stability ofthe hydrazone group of the linker, the linker can be varied by chemicalmodification, e.g., substitution, allowing tuning to achieve moreefficient release in the lysosome with a minimized loss in circulation.

Hydrazone-containing linkers can contain additional cleavage sites, suchas additional acid-labile cleavage sites and/or enzymatically labilecleavage sites. Conjugates including exemplary hydrazone-containinglinkers can include, for example, the following structures:

wherein D is an immune-stimulatory compound and Ab is an antibody orantigen-binding fragment thereof, respectively, and n represents thenumber of compound-bound linkers (LP) bound to the antibody orantigen-binding fragment thereof. In certain linkers, such as linker(Ia), the linker can comprise two cleavable groups, a disulfide and ahydrazone moiety. For such linkers, effective release of the unmodifiedfree immune-stimulatory compound can require acidic pH or disulfidereduction and acidic pH. Linkers such as (Ib) and (Ic) can be effectivewith a single hydrazone cleavage site.

Other acid-labile groups that can be included in linkers includecis-aconityl-containing linkers. cis-Aconityl chemistry can use acarboxylic acid juxtaposed to an amide bond to accelerate amidehydrolysis under acidic conditions.

Cleavable linkers can also include a disulfide group. Disulfides can bethermodynamically stable at physiological pH and can be designed torelease an immune-stimulatory compound upon internalization insidecells, wherein the cytosol can provide a significantly more reducingenvironment compared to the extracellular environment. Scission ofdisulfide bonds can require the presence of a cytoplasmic thiolcofactor, such as (reduced) glutathione (GSH), such thatdisulfide-containing linkers can be reasonably stable in circulation,selectively releasing the immune-stimulatory compound in the cytosol.The intracellular enzyme protein disulfide isomerase, or similar enzymescapable of cleaving disulfide bonds, can also contribute to thepreferential cleavage of disulfide bonds inside cells. GSH can bepresent in cells in the concentration range of 0.5-10 mM compared with asignificantly lower concentration of GSH or cysteine, the most abundantlow-molecular weight thiol, in circulation at approximately 5 μM. Tumorcells, where irregular blood flow can lead to a hypoxic state, canresult in enhanced activity of reductive enzymes and therefore evenhigher glutathione concentrations. The in vivo stability of adisulfide-containing linker can be enhanced by chemical modification ofthe linker, e.g., use of steric hindrance adjacent to the disulfidebond.

Immune-stimulatory conjugates including disulfide-containing linkers caninclude the following structures:

wherein D is an immune-stimulatory compound and Ab is an antibody orantigen-binding fragment thereof, respectively, n represents the numberof compounds bound to linkers bound to the antibody or antigen-bindingfragment thereof and R is independently selected at each occurrence fromhydrogen or alkyl, for example. Increasing steric hindrance adjacent tothe disulfide bond can increase the stability of the linker. Structuressuch as (IIa) and (IIe) can show increased in vivo stability when one ormore R groups is selected from a lower alkyl such as methyl.

Another type of linker that can be used is a linker that is specificallycleaved by an enzyme. For example, the linker can be cleaved by alysosomal enzyme. Such linkers can be peptide-based or can includepeptidic regions that can act as substrates for enzymes. Peptide basedlinkers can be more stable in plasma and extracellular milieu thanchemically labile linkers.

Peptide bonds can have good serum stability, as lysosomal proteolyticenzymes can have very low activity in blood due to endogenous inhibitorsand the unfavorable pH value of blood compared to lysosomes. Release ofan immune-stimulatory compound from an antibody or antigen-bindingfragment thereof can occur due to the action of lysosomal proteases,e.g., cathepsin and plasmin. These proteases can be present at elevatedlevels in certain tumor tissues. A linker can be cleavable by alysosomal enzyme. The lysosomal enzyme can be, for example, cathepsin B,cathepsin S, β-glucuronidase, or β-galactosidase.

The cleavable peptide can be selected from tetrapeptides such asGly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, dipeptides such as Val-Cit, Val-Ala,and Phe-Lys, or other peptides. Dipeptides can have lower hydrophobicitycompared to longer peptides, depending on the composition of thepeptide.

A variety of dipeptide-based cleavable linkers can be used in theimmune-stimulatory conjugates described herein.

Enzymatically cleavable linkers can include a self-immolative spacer tospatially separate the immune-stimulatory compound from the site ofenzymatic cleavage. The direct attachment of an immune-stimulatorycompound to a peptide linker can result in proteolytic release of theimmune-stimulatory compound or of an amino acid adduct of theimmune-stimulatory compound, thereby impairing its activity. The use ofa self-immolative spacer can allow for the elimination of the fullyactive, chemically unmodified immune-stimulatory compound upon amidebond hydrolysis.

One self-immolative spacer can be a bifunctional para-aminobcnzylalcohol group (PABA), which can link to the peptide through the aminogroup, forming an amide bond, while amine containing immune-stimulatorycompounds can be attached through carbamate functionalities to thebenzylic hydroxyl group of the linker (to give a p-amidobenzylcarbamate,PABC). The resulting pro-immune-stimulatory compound can be activatedupon protease-mediated cleavage, leading to a 1,6-elimination reactionreleasing the unmodified immune-stimulatory compound, carbon dioxide,and remnants of the linker. The following scheme depicts thefragmentation of p-amidobenzyl carbamate and release of theimmune-stimulatory compound:

wherein X-D represents the unmodified immune-stimulatory compound andthe carbonyl group adjacent “peptide” is part of the peptide.Heterocyclic variants of this self-immolative group have also beendescribed.

An enzymatically cleavable linker can be a ß-glucuronic acid-basedlinker. Facile release of an immune-stimulatory compound can be realizedthrough cleavage of the ß-glucuronide glycosidic bond by the lysosomalenzyme ß-glucuronidase. This enzyme can be abundantly present withinlysosomes and can be overexpressed in some tumor types, while the enzymeactivity outside cells can be low. ß-Glucuronic acid-based linkers canbe used to circumvent the tendency of an immune-stimulatory conjugate toundergo aggregation due to the hydrophilic nature of ß-glucuronides. Incertain embodiments, ß-glucuronic acid-based linkers can link anantibody or antigen-binding fragment thereof to a hydrophobicimmune-stimulatory compound. The following scheme depicts the release ofan immune-stimulatory compound (D) from an immune-stimulatory conjugatecontaining a β-glucuronic acid-based linker shown below, wherein Abindicates the antibody or antigen-binding fragment thereof.

A variety of cleavable ß-glucuronic acid-based linkers useful forlinking drugs such as auristatins, camptothecin and doxorubicinanalogues, CBI minor-groove binders, and psymberin to antibodies havebeen described. These ß-glucuronic acid-based linkers may be used in theconjugates described herein. In certain embodiments, the enzymaticallycleavable linker is a ß-galactoside-based linker. ß-Galactoside ispresent abundantly within lysosomes, while the enzyme activity outsidecells is low.

Additionally, immune-stimulatory compounds containing a phenol group canbe covalently bonded to a linker through the phenolic oxygen. One suchlinker relies on a methodology in which a diamino-ethane “Space Link” isused in conjunction with traditional “PABO”-based self-immolative groupsto deliver phenols.

Cleavable linkers can include non-cleavable portions or segments, and/orcleavable segments or portions can be included in an otherwisenon-cleavable linker to render it cleavable. By way of example only,polyethylene glycol (PEG) and related polymers can include cleavablegroups in the polymer backbone. For example, a polyethylene glycol orpolymer linker can include one or more cleavable groups such as adisulfide, a hydrazone or a dipeptide.

Other degradable linkages that can be included in linkers can includeester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on animmune-stimulatory compound, wherein such ester groups can hydrolyzeunder physiological conditions to release the immune-stimulatorycompound. Hydrolytically degradable linkages can include, but are notlimited to, carbonate linkages; imine linkages resulting from reactionof an amine and an aldehyde; phosphate ester linkages formed by reactingan alcohol with a phosphate group; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; and oligonucleotidelinkages formed by a phosphoramidite group, including but not limitedto, at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide.

A linker can contain an enzymatically cleavable peptide moiety, forexample, a linker comprising structural formula (IIIa), (IIIb), (IIIc),or (IIId):

or a pharmaceutically acceptable salt thereof, wherein: “peptide”represents a peptide (illustrated in N→C orientation, wherein peptideincludes the amino and carboxy “termini”) that is cleavable by alysosomal enzyme; T represents a polymer comprising one or more ethyleneglycol units or an alkylene chain, or combinations thereof; R^(a) isselected from hydrogen, alkyl, sulfonate and methyl sulfonate; R^(y) ishydrogen or C₁₋₄ alkyl-(O)_(r)—(C₁₋₄ alkylene)_(s)-G¹ or C₁₋₄alkyl-(N)—[(C₁₋₄ alkylene)-G¹]₂; R^(z) is C₁₋₄ alkyl-(O)_(r)—(C₁₋₄alkylene)_(s)-G²; G¹ is SO₃H, CO₂H, PEG 4-32, or a sugar moiety; G² isSO₃H, CO₂H, or a PEG 4-32 moiety; r is 0 or 1; s is 0 or 1; p is aninteger ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to animmune-stimulatory compound; and * represents the point of attachment tothe remainder of the linker.

In certain embodiments, the peptide can be selected from natural aminoacids, unnatural amino acids or combinations thereof. In certainembodiments, the peptide can be selected from a tripeptide or adipeptide. In particular embodiments, the dipeptide can comprise L-aminoacids and be selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala;Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit;Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys;Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit;Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit, or salts thereof.

Exemplary embodiments of linkers according to structural formula (Ilia)are illustrated below (as illustrated, the linkers include a reactivegroup suitable for covalently linking the linker to an antibody orantigen-binding fragment thereof):

wherein

indicates an attachment site of a linker to an immune-stimulatorycompound.

Exemplary embodiments of linkers according to structural formula (IIIb),(IIIc), or (IIId) that can be included in the conjugates describedherein can include the linkers illustrated below (as illustrated, thelinkers can include a reactive group suitable for covalently linking thelinker to an antibody or antigen-binding fragment thereof):

wherein

indicates an attachment site to an immune-stimulatory compound

The linker can contain an enzymatically cleavable sugar moiety, forexample, a linker comprising structural formula (IVa), (IVb), (IVc),(IVd), or (IVe):

or a pharmaceutically acceptable salt thereof, wherein: q is 0 or 1; ris 0 or 1; X¹ is CH₂, O or NH;

represents the point of attachment of the linker to animmune-stimulatory compound; and * represents the point of attachment tothe remainder of the linker.

Exemplary embodiments of linkers according to structural formula (IVa)that may be included in the immune-stimulatory conjugates describedherein can include the linkers illustrated below (as illustrated, thelinkers include a group suitable for covalently linking the linker to anantibody or antigen-binding fragment thereof):

wherein

represents the point of attachment of a linker to an immune-stimulatory.

Exemplary embodiments of linkers according to structural formula (IVb)that may be included in the conjugates described herein include thelinkers illustrated below (as illustrated, the linkers include a groupsuitable for covalently linking the linker to an antibody orantigen-binding fragment thereof):

wherein

represents the point of attachment of a linker to an immune-stimulatorycompound.

Exemplary embodiments of linkers according to structural formula (IVc)that may be included in the conjugates described herein include thelinkers illustrated below (as illustrated, the linkers include a groupsuitable for covalently linking the linker to an antibody orantigen-binding fragment thereof):

wherein

represents the point of attachment of a linker to an immune-stimulatorycompound.

Exemplary embodiments of linkers according to structural formula (IVd)that may be included in the conjugates described herein include thelinkers illustrated below (as illustrated, the linkers include a groupsuitable for covalently linking the linker to an antibody orantigen-binding fragment thereof):

wherein

represents the point of attachment of a linker to an immune-stimulatorycompound.

Exemplary embodiments of linkers according to structural formula (IVe)that may be included in the conjugates described herein include thelinkers illustrated below (as illustrated, the linkers include a groupsuitable for covalently linking the linker to an antibody orantigen-binding fragment thereof):

wherein

represents the point of attachment of a linker to an immune-stimulatorycompound.

Although cleavable linkers can provide certain advantages, the linkerscomprising the conjugate described herein need not be cleavable. Fornon-cleavable linkers, the immune-stimulatory compound release may notdepend on the differential properties between the plasma and somecytoplasmic compartments. The release of the immune-stimulatory compoundcan occur after internalization of the immune-stimulatory conjugate viaantigen-mediated endocytosis and delivery to lysosomal compartment,where the antibody or antigen-binding fragment thereof can be degradedto the level of amino acids through intracellular proteolyticdegradation. This process can release an immune-stimulatory compoundderivative, which is formed by the immune-stimulatory compound, thelinker, and the amino acid residue or residues to which the linker wascovalently attached. The immune-stimulatory compound derivative fromimmune-stimulatory conjugates with non-cleavable linkers can be morehydrophilic and less membrane permeable, which can lead to lessbystander effects and less nonspecific toxicities compared toimmune-stimulatory conjugates with a cleavable linker.Immune-stimulatory conjugates with non-cleavable linkers can havegreater stability in circulation than immune-stimulatory conjugates withcleavable linkers. Non-cleavable linkers can include alkylene chains, orcan be polymeric, such as, for example, based upon polyalkylene glycolpolymers, amide polymers, or can include segments of alkylene chains,polyalkylene glycols and/or amide polymers. The linker can contain apolyethylene glycol segment having from 1 to 6 ethylene glycol units.

The linker can be non-cleavable in vivo, for example, a linker accordingto the formulations below:

or salts thereof, wherein: R^(a) is selected from hydrogen, alkyl,sulfonate and methyl sulfonate; R^(x) is a reactive moiety including afunctional group capable of covalently linking the linker to an antibodyor antigen-binding fragment thereof; and

represents the point of attachment of the linker to animmune-stimulatory compound.

Exemplary embodiments of linkers according to structural formula(Va)-(Vf) that may be included in the conjugates described hereininclude the linkers illustrated below (as illustrated, the linkersinclude a group suitable for covalently linking the linker to anantibody or antigen-binding fragment thereof, and

represents the point of attachment of the linker to animmune-stimulatory compound:

In some embodiments, a linker is represented by formula (V):

wherein:

L⁴ represents the C-terminus of the peptide;

L⁵ is selected from a bond, alkylene and heteroalkylene, wherein L⁵ isoptionally substituted with one or more groups independently selectedfrom R³²;

RX* comprises a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of a polypeptide, such as an antibody, wherein

on RX* represents the point of attachment to the residue of thepolypeptide, such as the antibody, and the other

represents the point of attachment to the myeloid cell agonist, such asa TLR8 or TLR7 agonist; and

R³² is independently selected at each occurrence from halogen, —OH, —CN,—O— alkyl, —SH, ═O, ═S, —NH₂, —NO₂; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, andC₂₋₁₀ alkynyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —OH, —CN, —O-alkyl,—SH, ═O, ═S, —NH₂, and —NO₂.

Attachment groups that are used to attach the linkers to an antibody orantigen-binding fragment thereof can be electrophilic in nature andinclude, for example, maleimide groups, alkynes, alkynoates, allenes andallenoates, activated disulfides, active esters such as NHS esters andHOBt esters, haloformates, acid halides, alkyl, and benzyl halides suchas haloacetamides. There are also emerging technologies related to“self-stabilizing” maleimides and “bridging disulfides” that can be usedin accordance with the disclosure.

Maleimide groups are frequently used in the preparation of conjugatesbecause of their specificity for reacting with thiol groups of, forexample, cysteine groups of the antibody or antigen-binding fragmentthereof of a conjugate. The reaction between a thiol group of anantibody or antigen-binding fragment thereof and a drug with a linkerincluding a maleimide group proceeds according to the following scheme:

The reverse reaction leading to maleimide elimination from athio-substituted succinimide may also take place. This reverse reactionis undesirable as the maleimide group may subsequently react withanother available thiol group such as other proteins in the body havingavailable cysteines. Accordingly, the reverse reaction can undermine thespecificity of a conjugate. One method of preventing the reversereaction is to incorporate a basic group into the linking group shown inthe scheme above. Without wishing to be bound by theory, the presence ofthe basic group may increase the nucleophilicity of nearby watermolecules to promote ring-opening hydrolysis of the succinimide group.The hydrolyzed form of the attachment group is resistant todeconjugation in the presence of plasma proteins. So-called“self-stabilizing” linkers provide conjugates with improved stability. Arepresentative schematic is shown below:

The hydrolysis reaction schematically represented above may occur ateither carbonyl group of the succinimide group. Accordingly, twopossible isomers may result, as shown below:

The identity of the base as well as the distance between the base andthe maleimide group can be modified to tune the rate of hydrolysis ofthe thio-substituted succinimide group and optimize the delivery of aconjugate to a target by, for example, improving the specificity andstability of the conjugate.

Bases suitable for inclusion in a linker described herein, e.g., anylinker described herein with a maleimide group prior to conjugating toan antibody or antigen-binding fragment thereof, may facilitatehydrolysis of a nearby succinimide group formed after conjugation of theantibody or antigen-binding fragment thereof to the linker. Bases mayinclude, for example, amines (e.g., —N(R²⁶)(R²⁷), where R²⁶ and R²⁷ areindependently selected from H and C₁₋₆ alkyl), nitrogen-containingheterocycles (e.g., a 3- to 12-membered heterocycle including one ormore nitrogen atoms and optionally one or more double bonds), amidines,guanidines, and carbocycles or heterocycles substituted with one or moreamine groups (e.g., a 3- to 12-membered aromatic or non-aromatic cycleoptionally including a heteroatom such as a nitrogen atom andsubstituted with one or more amines of the type —N(R²⁶)(R²⁷), where R²⁶and R²⁷ are independently selected from H or C₁₋₆ alkyl). A basic unitmay be separated from a maleimide group by, for example, an alkylenechain of the form —(CH₂)_(m)—, where m is an integer from 0 to 10. Analkylene chain may be optionally substituted with other functionalgroups as described herein.

A linker described herein with a maleimide group may include an electronwithdrawing group such as, but not limited to, —C(O)R, ═O, —CN, —NO₂,—CX₃, —X, —COOR, —CONR₂, —COR, —COX, —SO₂R, —SO₂OR, —SO₂NHR, —SO₂NR₂,PO₃R₂, —P(O)(CH₃)NHR, —NO, —NR₃ ⁺, —CR═CR₂, and —C═CR, where each R isindependently selected from H and C₁₋₆ alkyl and each X is independentlyselected from F, Br, Cl, and I. Self-stabilizing linkers may alsoinclude aryl, e.g., phenyl, or heteroaryl, e.g., pyridine, groupsoptionally substituted with electron withdrawing groups such as thosedescribed herein.

Examples of self-stabilizing linkers are provided in, e.g., U.S. PatentPublication No. US 2013/0309256, the linkers of which are incorporatedby reference herein. It will be understood that a self-stabilizinglinker useful in conjunction with immune-stimulatory compounds may beequivalently described as unsubstituted maleimide-including linkers,thio-substituted succinimide-including linkers, or hydrolyzed,ring-opened thio-substituted succinimide-including linkers.

In certain embodiments, a linker comprises a stabilizing linker moietyselected from:

In the scheme provided above, the bottom structure may be referred to as(maleimido)-DPR-Val-Cit-PAB, where DPR refers to diaminopropinoic acid,Val refers to valine, Cit refers to citrulline, and PAB refers topara-aminobenzylcarbonyl.

represents the point of attachment to an immune-stimulatory compound.

A method for bridging a pair of sulfhydryl groups derived from reductionof a native hinge disulfide bond has been disclosed and is depicted inthe schematic below. An advantage of this methodology is the ability tosynthesize homogenous DAR4 conjugates by full reduction of IgGs (to give4 pairs of sulfhydryls from interchain disulfides) followed by reactionwith 4 equivalents of the alkylating agent. Conjugates containing“bridged disulfides” are also claimed to have increased stability.

Similarly, as depicted below, a maleimide derivative that is capable ofbridging a pair of sulfhydryl groups has been developed.

A linker can contain the following structural formulas (VIa), (VIb), or(VIc):

or salts thereof, wherein: R^(q) is H or —O—(CH₂CH₂O)₁₁—CH₃; x is 0 or1; y is 0 or 1; G² is —CH₂CH₂CH₂SO₃H or —CH₂CH₂O—(CH₂CH₂O)₁₁—CH₃; R^(w)is —O—CH₂CH₂SO₃H or —NH(CO)—CH₂CH₂O—(CH₂CH₂O)₁₂—CH₃; and * representsthe point of attachment to the remainder of the linker.

Exemplary embodiments of linkers according to structural formula (VIa)and (VIb) that can be included in the conjugates described herein caninclude the linkers illustrated below (as illustrated, the linkers caninclude a group suitable for covalently linking the linker to anantibody or antigen-binding fragment thereof):

wherein

N represents the point of attachment of the linker to animmune-stimulatory compound.

Exemplary embodiments of linkers according to structural formula (Vic)that can be included in the immune-stimulatory conjugates describedherein can include the linkers illustrated below (as illustrated, thelinkers can include a group suitable for covalently linking the linkerto an antibody or antigen-binding fragment thereof):

wherein

represents the point of attachment of the linker to animmune-stimulatory compound.

A linker can be attached to an antibody or antigen-binding fragmentthereof at any suitable position. Factors to be considered in selectingan attachment site include whether the linker is cleavable ornon-cleavable, the reactive group of the linker for attachment to theantibody or antigen-binding fragment thereof, the chemical nature of theimmune-stimulatory compound and compatibility with reactive sites on thelinker and the antibody or antigen-binding fragment thereof, and theeffect of the attachment site on functional activities of the Fc domain.A linker may be attached to a terminus of an amino acid sequence of anantibody or antigen-binding fragment thereof or can be attached to aside chain of an amino acid of an antibody or antigen-binding fragmentthereof, such as the side chain of a lysine, serine, threonine,cysteine, tyrosine, aspartic acid, glutamine, a non-natural amino acidresidue, or glutamic acid residue. A linker may be bound to a terminusof an amino acid sequence of an Fc domain or Fc region of an antibody orantigen-binding fragment thereof, or may be bound to a side chain of anamino acid of an Fc domain of an antibody or antigen-binding fragmentthereof, such as the side chain of a lysine, serine, threonine,cysteine, tyrosine, aspartic acid, glutamine, a non-natural amino acidresidue, or glutamic acid residue.

In some embodiments, a linker is attached to a hinge cysteine of anantibody Fc domain. A linker can be attached to an antibody orantigen-binding fragment thereof at a light chain constant domainlysine. A linker can be attached to an antibody or antigen-bindingfragment thereof at an engineered cysteine in the light chain. A linkercan be attached to an antibody or antigen-binding fragment thereof at anengineered light chain glutamine. A linker can be attached to anantibody or antigen-binding fragment thereof at an unnatural amino acidengineered into the light chain. A linker can be attached to an antibodyor antigen-binding fragment thereof at a heavy chain constant domainlysine. A linker can be attached to an antibody or antigen-bindingfragment thereof at an engineered cysteine in the heavy chain. A linkercan be attached to an antibody or antigen-binding fragment thereof at anengineered heavy chain glutamine. A linker can be attached to anantibody or antigen-binding fragment thereof at an unnatural amino acidengineered into the heavy chain. Amino acids can be engineered into anamino acid sequence of an antibody or antigen-binding fragment thereofas described herein or as known to the skilled artisan and can beconnected to a linker of a conjugate. Engineered amino acids can beadded to a sequence of existing amino acids. Engineered amino acids canbe substituted for one or more existing amino acids of a sequence ofamino acids.

A linker can be attached to an antibody or antigen-binding fragmentthereof via a sulfhydryl group. A linker can be attached to an antibodyor antigen-binding fragment thereof via a primary amine. A linker can bea link created between an unnatural amino acid on an antibody byreacting with oxime bond that was formed by modifying a ketone groupwith an alkoxyamine on an immune stimulatory compound.

As is known by skilled artisans, the linker selected for a particularconjugate may be influenced by a variety of factors, including but notlimited to, the site of attachment to the antibody or antigen-bindingfragment thereof (e.g., lys, cys or other amino acid residues),structural constraints of the drug pharmacophore and the lipophilicityof the drug. The specific linker selected for a conjugate should seek tobalance these different factors for the specific antibody/drugcombination.

For example, conjugates have been observed to effect killing ofbystander antigen-negative cells present in the vicinity of theantigen-positive tumor cells. The mechanism of bystander cell killing byconjugates has indicated that metabolic products formed duringintracellular processing of the conjugates may play a role. Neutralcytotoxic metabolites generated by metabolism of the conjugates inantigen-positive cells appear to play a role in bystander cell killingwhile charged metabolites may be prevented from diffusing across themembrane into the medium, or from the medium across the membrane, andtherefore cannot affect bystander killing. In certain embodiments, thelinker is selected to attenuate the bystander effect caused by cellularmetabolites of the conjugate. In certain embodiments, the linker isselected to increase the bystander effect.

The properties of the linker, or linker-compound, may also impactaggregation of the conjugate under conditions of use and/or storage.Typically, conjugates reported in the literature contain no more than3-4 drug molecules per antibody molecule. Attempts to obtain higherdrug-to-antibody ratios (“DAR”) often failed, particularly if both thedrug and the linker were hydrophobic, due to aggregation of theconjugate. In many instances, DARs higher than 3-4 could be beneficialas a means of increasing potency. In instances where animmune-stimulatory compound is more hydrophobic in nature, it may bedesirable to select linkers that are relatively hydrophilic as a meansof reducing conjugate aggregation, especially in instances where DARsgreater than 3-4 are desired. Thus, in certain embodiments, a linkerincorporates chemical moieties that reduce aggregation of the conjugatesduring storage and/or use. A linker may incorporate polar or hydrophilicgroups such as charged groups or groups that become charged underphysiological pH to reduce the aggregation of the conjugates. Forexample, a linker may incorporate charged groups such as salts or groupsthat deprotonate, e.g., carboxylates, or protonate, e.g., amines, atphysiological pH.

In particular embodiments, the aggregation of the conjugates duringstorage or use is less than about 40% as determined by size-exclusionchromatography (SEC). In particular embodiments, the aggregation of theconjugates during storage or use is less than 35%, such as less thanabout 30%, such as less than about 25%, such as less than about 20%,such as less than about 15%, such as less than about 10%, such as lessthan about 5%, such as less than about 4%, or even less, as determinedby size-exclusion chromatography (SEC).

Conjugates

A conjugate as described herein comprises an anti-Nectin-4 antibody oran antigen-binding fragment thereof and at least one linker attached toat least one immune-stimulatory compound, such as a myeloid cell agonistor other agonist (e.g., TLR8 agonist, TLR7 agonist, other TLR agonist,STING agonist, RIG-I-Like receptor agonist, c-type lectin receptorsagonist, or cytosolic DNA Sensors agonist). In some aspects, the presentdisclosure provides a conjugate represented by Formula I:

wherein. A is the anti-Nectin-4 antibody or an antigen-binding fragmentthereof, L is the linker; D_(x) is the immune-stimulatory compound; n isselected from 1 to 20; and z is selected from 1 to 20.

In some embodiments, the immune-stimulatory compound is a myeloid cellagonist. In some embodiments, the immune-stimulatory compound is a TLR8agonist. In some embodiments, the immune-stimulatory compound is a TLR7agonist. In some embodiments, the immune-stimulatory compound is a TLR3agonist. In some embodiments, the immune-stimulatory compound is a TLR4agonist. In some embodiments, the immune-stimulatory compound is a TLR5agonist. In some embodiments, the immune-stimulatory compound is a TLR9agonist. In some embodiments, the immune-stimulatory compound is a STINGagonist. Exemplary STING agonist compounds include RG7854, ADU-S100,MK-1454, MK-2118, BMS-986301, GSK3745417, SB-11285, and IMSA-101. Insome embodiments, the immune-stimulatory compound is a RIG-I-Likereceptor agonist. In some embodiments, the immune-stimulatory compoundis a c-type lectin receptors agonist. In some embodiments, theimmune-stimulatory compound is a cytosolic DNA Sensors agonist.

In some aspects, the present disclosure provides a conjugate comprisingat least one immune-stimulatory compound (e.g., a compound or saltthereof), an anti-Nectin-4 antibody or an antigen-binding fragmentthereof, and at least one linker, wherein each immune-stimulatorycompound is linked, i.e., covalently bound, to the anti-Nectin-4antibody or an antigen-binding fragment thereof through a linker. Thelinker can be selected from a cleavable or non-cleavable linker. In someembodiments, the linker is cleavable. In other embodiments, the linkeris non-cleavable. Linkers are further described in the presentapplication in the preceeding section, any one of which can be used toconnect an antibody or antigen-binding fragment thereof to animmune-stimulatory compound.

In a conjugate, the drug loading is represented by z, the number ofimmune-stimulatory compound-linker molecules per antibody, or the numberof immune-stimulatory compounds per antibody, depending on theparticular conjugate. Depending on the context, z can represent theaverage number of immune-stimulatory compound(-linker) molecules perantibody, also referred to the average drug loading, z can range from 1to 20, from 1-50 or from 1-100. In some conjugates, z is preferably from1 to 8. In some preferred embodiments, when z represents the averagedrug loading, z ranges from about 2 to about 5. In some embodiments, zis about 2, about 3, about 4, or about 5. The average number ofimmune-stimulatory compounds per antibody in a preparation of conjugatemay be characterized by conventional means such as mass spectroscopy,liquid chromatography/mass spectrometry (LC/MS), HIC, ELISA assay, andHPLC.

A number of conjugates are consistent with the disclosure herein. Theconjugates generally comprise an immune-stimulatory compound covalentlybound to an anti-Nectin-4 antibody or an antigen-binding fragmentthereof that localizes the conjugate to a target tissue, cell populationor cell. The anti-Nectin-4 antibody or an antigen-binding fragmentthereof is covalently attached to each immune-stimulatory compound,either directly or through a linker that tethers the immune-stimulatorycompound to the anti-Nectin-4 antibody or an antigen-binding fragmentthereof. Anti-Nectin-4 antibodies or an antigen-binding fragmentsthereof listed herein as well as are consistent with the conjugates asdisclosed herein.

Some exemplary conjugates are as follows. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one immune-stimulatory compound, and optionally atleast one linker. A conjugate can comprise an anti-Nectin-4 antibody oran antigen-binding fragment thereof of this disclosure, at least oneTLR7 agonist, and at least one linker. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one TLR8 agonist, and at least one linker. Aconjugate can comprise an anti-Nectin-4 antibody or an antigen-bindingfragment thereof of this disclosure, at least one compound of Category A(TLR8 agonists), and at least one linker. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one compound of Category B (TLR7 agonists), and atleast one linker. A conjugate can comprise an anti-Nectin-4 antibody oran antigen-binding fragment thereof of this disclosure, at least onecompound of Category C (TLR8 agonists), and at least one linker. Aconjugate can comprise an anti-Nectin-4 antibody or an antigen-bindingfragment thereof of this disclosure, at least one TLR3 agonist, and atleast one linker. A conjugate can comprise an anti-Nectin-4 antibody oran antigen-binding fragment thereof of this disclosure, at least oneTLR4 agonist, and at least one linker. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one TLR5 agonist, and at least one linker. Aconjugate can comprise an anti-Nectin-4 antibody or an antigen-bindingfragment thereof of this disclosure, at least one TLR9 agonist, and atleast one linker. A conjugate can comprise an anti-Nectin-4 antibody oran antigen-binding fragment thereof of this disclosure, at least oneSTING agonist, and at least one linker. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one RIG-I agonist, and at least one linker. Aconjugate can comprise an anti-Nectin-4 antibody or an antigen-bindingfragment thereof of this disclosure, at least one c-type lectin receptoragonist, and at least one linker. A conjugate can comprise ananti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure, at least one cytosolic DNA Sensors agonist, and at least onelinker.

Exemplary Conjugates

In certain embodiments, the disclosure provides an immune-stimulatoryconjugate (or conjugate) of an anti-Nectin-4 antibody or anantigen-binding fragment thereof and at least one compound of any one ofCategory A Formulas (IA), (IB), (IIA), (IIB), (IIC), (IIIA), (IIIB),(IVA), (IVB), and (IVC), each compound optionally attached to theantibody or an antigen-binding fragment via a linker. In certainembodiments, the disclosure provides an immune-stimulatory conjugate ofan anti-Nectin-4 antibody or an antigen-binding fragment thereof and atleast one compound of any one of Category B Formulas (IA), (IB), (IC),(IIA), (IIB), and (IIC), each compound optionally attached to theantibody or an antigen-binding fragment via a linker. In certainembodiments, the disclosure provides an immune-stimulatory conjugate ofan anti-Nectin-4 antibody or an antigen-binding fragment thereof of thisdisclosure and at least one compound of any one of Category C FormulasIa-Ih each compound optionally attached to the antibody or anantigen-binding fragment via a linker. In certain embodiments, theaverage Drug-to-Antibody Ratio (DAR) of the pharmaceutical compositioncomprising a conjugate of an anti-Nectin-4 antibody or anantigen-binding fragment thereof of this disclosure is selected from 1to about 8, 2 to about 6, about 3 to about 5, or about 4.

In certain embodiments, the disclosure provides a pharmaceuticalcomposition suitable for intravenous or subcutaneous administration,comprising an immune stimulatory compound of any one of Category AFormulas (IA), (IB), (IIA), (IIB), (IIC), (IIIA), (IIIB), (IVA), (IVB),and (IVC) conjugated to an anti-Nectin-4 antibody or an antigen-bindingfragment thereof of this disclosure, and a pharmaceutically acceptableexcipient. In certain embodiments, the disclosure provides apharmaceutical composition suitable for intravenous or subcutaneousadministration, comprising an immune stimulatory compound of any one ofCategory B Formulas (IA), (IB), (IC), (IIA), (IIB), and (IIC) conjugatedto an anti-Nectin-4 antibody or an antigen-binding fragment thereof ofthis disclosure, and a pharmaceutically acceptable excipient. In certainembodiments, the disclosure provides a pharmaceutical compositionsuitable for intravenous or subcutaneous administration, comprising animmune stimulatory compound of any one of Category C Formulas Ia-Ihconjugated to an anti-Nectin-4 antibody or an antigen-binding fragmentthereof of this disclosure, and a pharmaceutically acceptable excipient.In certain embodiments, the average Drug-to-Antibody Ratio (DAR) of apharmaceutical composition of the aforementioned conjugates is selectedfrom 1 to about 8, 2 to about 6, about 3 to about 5, or about 4.

In certain embodiments, the disclosure provides a method for thetreatment of a disease treatable by a TLR agonist (e.g., cancer)comprising subcutaneously administering an effective amount of aconjugate of a compound of any one of Category A Formulas (IA), (IB),(IIA), (IIB), (IIC), (IIIA), (IIIB), (IVA), (IVB), and (IVC), or apharmaceutical composition thereof suitable for intravenous orsubcutaneous administration to a subject in need thereof. In certainembodiments, the disclosure provides a method for the treatment ofcancer (e.g., bladder, breast, lung, head and neck, cervical),comprising intravenously or subcutaneously administering an effectiveamount of the conjugate of a compound of any one of Category B Formulas(I A), (IB), (IC), (IIA), (IIB), and (IIC), or a pharmaceuticalcomposition thereof suitable for subcutaneous administration to asubject in need thereof. In certain embodiments, the disclosure providesa method for the treatment of cancer (e.g., bladder, breast, lung, headand neck, cervical), comprising intravenously or subcutaneouslyadministering an effective amount of the conjugate of a compound of anyone of Category C Formulas Ia-Ih, or a pharmaceutical compositionthereof suitable for subcutaneous administration to a subject in needthereof. In any of the embodiments herein, the conjugate may beadministered by slow infusion.

The disclosure provides a method of preparing an antibody conjugate ofthe formula:

wherein:

n is selected from 1 to 20;

L³ is a linker; and

D is selected from a compound or salt of a compound of any one ofCategory A Formulas (IA), (IB), (IIA), (IIB), (IIC), (IIIA), (IIIB),(IVA), (IVB), and (IVC); Category B Formulas (IA), (IB), (IC), (IIA),(IIB), and (IIC); and Category C Formulas (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig) and (Ih),

comprising contacting D-L³ with an anti-Nectin-4 antibody or anantigen-binding fragment thereof.

The disclosure provides a method of preparing an anti-Nectin-4 antibodyor an antigen-binding fragment thereof conjugate of the formula:

wherein: n is selected from 1 to 20; L³ is a linker; and D is selectedfrom a compound of any one of Category A Formulas (IA), (IB), (IIA),(IIB), (IIC), (IIIA), (IIIB), (IVA), (IVB), and (IVC); Category BFormulas (IA), (IB), (IC), (IIA), (IIB), and (IIC); and Category CFormulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih), comprisingcontacting L³ with the anti-Nectin-4 antibody or an antigen-bindingfragment thereof to form L³-anti-Nectin-4 antibody or antigen-bindingfragment thereof and contacting L³-anti-Nectin-4 antibody or anantigen-binding fragment thereof with D to form the conjugate.

In certain embodiments, the present disclosure provides a myeloid cellagonist conjugate or salt thereof represented by the formula:

wherein Antibody is an anti-Nectin-4 antibody comprising:

-   -   (a) a light chain CDR1, CDR2 and CDR3 set forth in the light        chain variable region amino acid sequence of SEQ ID NO: 14 or        13, a light chain constant region comprising SEQ ID NO:20, heavy        chain CDR1, CDR2 and CDR3 set forth in the heavy chain variable        region amino acid sequence of SEQ ID NO: 10, and a heavy chain        constant region comprising SEQ ID NO: 18, or    -   (b) a light chain comprising the amino acid sequence of SEQ ID        NO:28 and a heavy chain comprising the amino acid sequence of        SEQ ID NO:24, or    -   (c) a light chain comprising the amino acid sequence of SEQ ID        NO:27 and a heavy chain comprising the amino acid sequence of        SEQ ID NO:24; and

L³-D is a linker-TLR8 agonist and has the structure:

wherein RX is a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of the antibody construct, wherein

on RX* represents the point of attachment to a cysteine residue of theantibody construct. In certain embodiments, L³-D has the structure:

In other embodiments, L³-D has the structure:

In still other embodiments, L³-D has the structure:

In certain such embodiments, RX* is a succinamide moiety or a hydrolyzedsuccinamide moiety.

The compounds disclosed herein, in some embodiments, are used indifferent enriched isotopic forms, e.g., enriched in the content of ²H,³H, ¹¹C, ¹³C and/or ¹⁴C. In particular embodiments, the compound isdeuterated in at least one position. Such deuterated forms can be madeby the procedure described in U.S. Pat. Nos. 5,846,514 and 6,334,997. Asdescribed in U.S. Pat. Nos. 5,846,514 and 6,334,997, deuteration canimprove the metabolic stability and or efficacy, thus increasing theduration of action of drugs.

Unless otherwise stated, structures depicted herein are intended toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of the present disclosure.

The compounds of the present disclosure optionally contain unnaturalproportions of atomic isotopes at one or more atoms that constitute suchcompounds.

For example, the compounds may be labeled with isotopes, such as forexample, deuterium (²H), tritium (³H), iodine-125 (¹²⁵I) or carbon-14(¹⁴C). Isotopic substitution with ²H, ¹¹C, ¹³C, ¹⁴C, ¹⁵C, ¹²N, ¹³N, ¹⁵N,¹⁶N, ¹⁶O, ¹⁷O, ¹⁴F, ¹⁵F, ¹⁶F, ¹⁷F, ¹⁸F, ³³S, ³⁴S, ³⁵S, ³⁶S, ³⁵Cl, ³⁷Cl,⁷⁹Br, ⁸¹Br, ¹²⁵I are all contemplated. All isotopic variations of thecompounds of the present disclosure, whether radioactive or not, areencompassed within the scope of the present disclosure.

In certain embodiments, the compounds disclosed herein have some or allof the ¹H atoms replaced with ²H atoms. The methods of synthesis fordeuterium-containing compounds are known in the art and include, by wayof non-limiting example only, the following synthetic methods.

Deuterium substituted compounds are synthesized using various methodssuch as described in: Dean, Dennis C.; Editor. Recent Advances in theSynthesis and Applications of Radiolabeled Compounds for Drug Discoveryand Development. [In: Curr. Pharm. Des., 2000; 6(10)] 2000, 110 pp;George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compoundsvia Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21;and Evans, E. Anthony. Synthesis of radiolabeled compounds, J.Radioanal. Chem., 1981, 64(1-2), 9-32.

Deuterated starting materials are readily available and are subjected tothe synthetic methods described herein to provide for the synthesis ofdeuterium-containing compounds. Large numbers of deuterium-containingreagents and building blocks are available commercially from chemicalvendors, such as Aldrich Chemical Co.

Compounds of the present disclosure also include crystalline andamorphous forms of those compounds, pharmaceutically acceptable salts,and active metabolites of these compounds having the same type ofactivity, including, for example, polymorphs, pseudopolymorphs,solvates, hydrates, unsolvated polymorphs (including anhydrates),conformational polymorphs, and amorphous forms of the compounds, as wellas mixtures thereof.

Included in the present disclosure are salts, particularlypharmaceutically acceptable salts, of the compounds described herein.The compounds of the present disclosure that possess a sufficientlyacidic, a sufficiently basic, or both functional groups, can react withany of a number of inorganic bases, and inorganic and organic acids, toform a salt. Alternatively, compounds that are inherently charged, suchas those with a quaternary nitrogen, can form a salt with an appropriatecounterion, e.g., a halide such as bromide, chloride, or fluoride.

The compounds described herein may in some cases exist as diastereomers,enantiomers, or other stereoisomeric forms. The compounds presentedherein include all diastereomeric, enantiomeric, and epimeric forms aswell as the appropriate mixtures thereof. Separation of stereoisomersmay be performed by chromatography or by forming diastereomers andseparating by recrystallization, or chromatography, or any combinationthereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers,Racemates and Resolutions”, John Wiley and Sons, Inc., 1981, hereinincorporated by reference for this disclosure). Stereoisomers may alsobe obtained by stereoselective synthesis.

The methods and compositions described herein include the use ofamorphous forms as well as crystalline forms (also known as polymorphs).The compounds described herein may be in the form of pharmaceuticallyacceptable salts. As well, active metabolites of these compounds havingthe same type of activity are included in the scope of the presentdisclosure. In addition, the compounds described herein can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

In certain embodiments, compounds or salts of the compounds describedherein may be prodrugs attached to anti-Nectin-4 antibodies to formconjugates. The term “prodrug” is intended to encompass compounds which,under physiologic conditions, are converted into active compounds, e.g.,TLR8 agonists, TLR7 agonists, other TLR agonists, STING agonist,RIG-I-Like receptor agonists, c-type lectin receptors agonists, orcytosolic DNA Sensors agonists. One method for making a prodrug is toinclude one or more selected moieties which are hydrolyzed or otherwisecleaved under physiologic conditions to reveal the desired molecule. Inother embodiments, the prodrug is converted by an enzymatic activity ofthe host animal such as specific target cells in the host animal.

Prodrug forms of the herein described compounds, wherein the prodrug ismetabolized in vivo to produce a compound described herein are includedwithin the scope of the claims. In some cases, some of theherein-described compounds may be a prodrug for another derivative oractive compound.

In certain embodiments, an immune-stimulatory compound, such as a TLR8agonist or TLR7 agonist, is modified as a prodrug with a masking group,such that the TLR8 agonist, TLR7 agonist or other agonist, has limitedactivity or is inactive until it reaches an environment where themasking group is removed to reveal the active compound. For example, aTLR8 agonist or TLR7 agonist can be covalently modified at an amineinvolved in binding to the active site of a TLR8 receptor such that thecompound is unable to bind the active site of the receptor in itsmodified (prodrug) form. In such an example, the masking group isremoved under physiological conditions, e.g., enzymatic or acidicconditions, specific to the site of delivery, e.g., intracellular orextracellular adjacent to target cells. Masking groups may be removedfrom the amine of the compound or salt described herein due to theaction of lysosomal proteases, e.g., cathepsin and plasmin. Theseproteases can be present at elevated levels in certain tumor tissues.The masking group may be removed by a lysosomal enzyme. The lysosomalenzyme can be, for example, cathepsin B, cathepsin S, β-glucuronidase,or β-galactosidase.

In certain embodiments, an amine masking group inhibits binding of theamine group of the compound with residues of a TLR8 receptor. The aminemasking group may be removable under physiological conditions within acell but remains covalently bound to the amine outside of a cell.Masking groups that may be used to inhibit or attenuate binding of anamine group of a compound with residues of a TLR8 receptor include, forexample, peptides and carbamates.

Synthetic chemistry transformations and methodologies useful insynthesizing the compounds described herein are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieser and M.Fieser, Fieser and Fieser's Reagents for Organic Synthesis (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (1995).

Exemplary TLR8 and TLR7 agonists are provided herein. In someembodiments, a myeloid cell agonist-linker compound (Linker-Payload) isselected from any of Linker-Payloads provided herein. Examples of TLR8agonist linker-compounds are provided in Table 2 and theirstereoisomers. Examples of TLR7 agonist linker-compounds are provided inTable 4 and their stereoisomers. It is understood that for the compoundsprovided in Tables 2 and 4, salts of the compounds are alsocontemplated.

TABLE 2 TLR8 agonist Linker-Compounds 2.1-2.22 Linker- Com- poundStructure 2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

2.10

2.11

2.12

2.14

2.15

2.16

2.17

2.20

2.21

2.22

TABLE 4 TLR7 Agonist Linker-Compounds 4.1-4.20 Linker- CompoundStructure 4.1

4-((S)-24(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2-yl)carbamate4.2

tert-butyl(2-((2-(((S)-1-((2-((1-(1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-5,5,11,11-tetrannethyl-3,6-dioxo-2,10-dioxa-4,7-diazadodecan-12-yl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate 4.3

N-(1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethypamino)-2-methyl-1-oxopropan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 4.4

N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-2-methylpropanamide4.5

N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide 4.6

N-(2-((1-(4-amino-2-(ethoxynnethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 4.7

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)carbamate4.8

(9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((1-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate4.9

tert-butyl (S)-(1-((1-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate4.10

tert-butyl((S)-1-((1-(2-(2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanannido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 4.11

tert-butyl(S)-2-((1-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamoyl)pyrrolidine-1-carboxylate4.12

tert-butyl (S)-2-((1-(2-(2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamoyl)pyrrolidine-1-carboxylate 4.13

4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanamido)benzyl(1-(2-(2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamate4.14

tert-butyl(S)-(2-((2-((1-((2-((1-(2-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-methylpropanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate4.15

tert-butyl(2-((1-(2-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-methylpropanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-2-oxoethyl)carbamate 4.16

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(14(24(1-(4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethypamino)-2-methyl-1-oxopropan-2-yl)carbamate4.17

(S)-N-(2-((1-(4-(2-amino-3-methylbutanannido)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide4.18

tert-butyl((S)-1-(((S)-1-((1-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate4.19

tert-butyl((S)-1-(((S)-1-((1-(2-(2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-mrthyl-1-oxobutan-2-yl)carbamate4.20

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-oxoethyl)carbamate

In some embodiments, a myeloid cell agonist conjugate compound isselected from:

and a salt of any one thereof, wherein the RX* is a bond, a succinimidemoiety, or a hydrolyzed succinimide moiety bound to a residue of ananti-Nectin-4 antibody construct, wherein

on RX* represents the point of attachment to the residue of theanti-Nectin-4 antibody construct.

The activation, stimulation or augmentation of an immune response by animmune-stimulatory conjugate, such as a myeloid cell agonist, can bemeasured in vitro by co-culturing immune cells (e.g., myeloid cells)with cells targeted by the conjugate and measuring cytokine release,chemokine release, proliferation of immune cells, upregulation of immunecell activation markers, and/or ADCC. ADCC can be measured bydetermining the percentage of remaining target cells in the co-cultureafter administration of the conjugate with the target cells, myeloidcells, and other immune cells. In some embodiments, animmune-stimulatory conjugate can activate or stimulate immune cellactivity, as determined by in vitro assay, such as a cytokine releaseassay, by detection of activation markers (e.g., MHC class II markers)or other assays known in the art. In some embodiments, animmune-stimulatory conjugate has an EC50 of 100 nM or less, as determineby cytokine release assay. In some embodiments, an immune-stimulatoryconjugate has an EC50 of 50 nM or less, as determine by cytokine releaseassay. In some embodiments, an immune-stimulatory conjugate has an EC50of 10 nM or less, as determine by cytokine release assay. In someembodiments, an immune-stimulatory conjugate has an EC50 of 1 mM orless.

Pharmaceutical Formulations

The conjugates described herein are useful as pharmaceuticalcompositions for administration to a subject in need thereof.Pharmaceutical compositions can comprise the conjugates described hereinand one or more pharmaceutically acceptable excipients, suitable foradministration by a selected route. A pharmaceutical composition cancomprise any conjugate described herein. A pharmaceutical compositioncan further comprise buffers, carbohydrates, and/or preservatives, asappropriate. Pharmaceutical compositions comprising a conjugate can bemanufactured, for example, by lyophilizing the conjugate, mixing,dissolving, emulsifying, encapsulating or entrapping the conjugate. Thepharmaceutical compositions can also include the conjugates describedherein in a free-base form or pharmaceutically-acceptable salt form.

Methods for formulation of the pharmaceutical compositions can includeformulating any of the conjugates described herein with one or moreinert, pharmaceutically-acceptable excipients or carriers to form asolid, semi-solid, or liquid composition for intravenous or subcutaneousadministration. Solid compositions can include, for example, powders,and in some aspects, the solid compositions further contain nontoxic,auxiliary substances, for example wetting or emulsifying agents, pHbuffering agents, and other pharmaceutically-acceptable additives.Alternatively, the compositions described herein can be lyophilized orin powder form for re-constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The pharmaceutical compositions and formulations can be sterilized.Sterilization can be accomplished by filtration through sterilefiltration.

The pharmaceutical compositions described herein can be formulated foradministration as an injection, e.g., an intravenous or subcutaneousinjection. Non-limiting examples of formulations for injection caninclude a sterile suspension, solution or emulsion in oily or aqueousvehicles. Suitable oily vehicles can include, but are not limited to,lipophilic solvents or vehicles such as fatty oils or synthetic fattyacid esters, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension. Thesuspension can also contain suitable stabilizers. Alternatively, thepharmaceutical compositions described herein can be lyophilized or inpowder form for reconstitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

The conjugates can be formulated for administration in a unit dosageform in association with a pharmaceutically acceptable vehicle. Suchvehicles can be inherently nontoxic, and non-therapeutic. A vehicle canbe water, saline, Ringer's solution, dextrose solution, and 5% humanserum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleatecan also be used. The vehicle can contain minor amounts of additivessuch as substances that enhance isotonicity and chemical stability(e.g., buffers and preservatives).

In some embodiments, an aqueous formulation of a conjugate providedherein, such as for subcutaneous administration, has a pH from 4-5.2.The aqueous formulation may comprise one or more excipients, such as,for example, one or more buffering agents, one or more lyoprotectants,and the like. In some embodiments, the pH of the formulation is from4-5.1, 4.1-5.1, 4.2-5.1, 4.3-5.1, 4.4-5.1, 4.5-5.1, 4-5, 4.1-5, 4.2-5,4.3-5, 4.4-5, or 4.5-5. In some embodiments, the formulation comprisesat least one buffer. In various embodiments, the buffer may be selectedfrom histidine, citrate, aspartate, acetate, phosphate, lactate,tromethamine, gluconate, glutamate, tartrate, succinate, malic acid,fumarate, α-ketoglutarate, and combinations thereof. In someembodiments, the buffer is at least one buffer selected from histidine,citrate, aspartate, acetate, and combinations thereof. In someembodiments, the buffer is a combination of histidine and aspartate. Insome embodiments, the total concentration of the buffer in the aqueousformulation is 10 mM to 40 mM, such as 15 mM-30 mM, 15 mM-25 mM, or 20mM.

In some embodiments, the aqueous formulation comprises at least onelyoprotectant. In some such embodiments, the at least one lyoprotectantis selected from sucrose, arginine, glycine, sorbitol, glycerol,trehalose, dextrose, alpha-cyclodextrin, hydroxypropylbeta-cyclodextrin, hydroxypropyl gamma-cyclodextrin, proline,methionine, albumin, mannitol, maltose, dextran, and combinationsthereof. In some embodiments, the lyoprotectant is sucrose. In someembodiments, the total concentration of lyoprotectant in the aqueousformulation is 3-12%, such as 5-12%, 6-10%, 5-9%, 7-9%, or 8%.

In some embodiments, the aqueous formulation comprises at least onesurfactant. Exemplary surfactants include polysorbate 80, polysorbate20, poloxamer 88, and combinations thereof. In some embodiments, theaqueous formulation comprises polysorbate 80. In some embodiments, thetotal concentration of the at least one surfactant is 0.01%-0.1%, suchas 0.01%-0.05%, 0.01%-0.08%, or 0.01%-0.06%, 0.01%-0.04%, 0.01%-0.03%,or 0.02%.

In some embodiments, the concentration of the conjugate in the aqueousformulation is 1 mg/mL-200 mg/mL, such as 10 mg/mL-160 mg/mL, 10mg/mL-140 mg/mL, 10 mg/mL-120 mg/mL, 20 mg/mL-120 mg/mL, or 30 mg/mL-120mg/mL, or 40 mg/mL-120 mg/mL, or 40 mg/mL-100 mg/mL. In someembodiments, the concentration of the conjugate in the aqueousformulation is 10 mg/mL-140 mg/mL or 40 mg/mL-140 mg/mL.

Therapeutic Applications

The antibodies or antigen-binding fragments thereof, conjugates andcompositions (e.g., pharmaceutical compositions) of the presentdisclosure can be useful for a plurality of different subjectsincluding, but are not limited to, a mammal, human, non-human mammal, adomesticated animal (e.g., laboratory animals, household pets, orlivestock), non-domesticated animal (e.g., wildlife), dog, cat, rodent,mouse, hamster, cow, bird, chicken, fish, pig, horse, goat, sheep,rabbit, and any combination thereof. In some embodiments, the subject isa human.

Anti-Nectin-4 antibodies or antigen-binding fragments thereof,conjugates, and compositions thereof of this disclosure can be useful asa therapeutic, for example, a treatment that can be administered to asubject in need thereof. A therapeutic effect of the antibodies,conjugates and compositions thereof of the present disclosure can beobtained in a subject by reduction, suppression, remission, oreradication of a disease state, including, but not limited to, a symptomthereof. A therapeutic effect in a subject having a disease or condition(e.g., cancer), or pre-disposed to have or is beginning to have thedisease or condition, can be obtained by a reduction, a suppression, aprevention, a remission, or an eradication of the condition or disease,or pre-condition or pre-disease state.

A “subject in need thereof” refers to an individual at risk of, orsuffering from, a disease, disorder or condition, such as cancer, thatis amenable to treatment or amelioration with an anti-Nectin-4 antibodyor antigen-binding fragment thereof, a conjugate of an anti-Nectin-4antibody or antigen-binding fragment thereof with a myeloid cell agonist(e.g., TLR8 agonist), or a composition thereof as provided herein. Incertain embodiments, a subject in need thereof is administered ananti-Nectin-4 antibody or antigen-binding fragment thereof, a conjugateof an anti-Nectin-4 antibody or antigen-binding fragment thereof with amyeloid cell agonist (e.g., TLR8 agonist), or a composition thereof asprovided herein to treat cancer, wherein the cancer comprises a tumorhaving excess Nectin-4 as compared to normal tissue.

In practicing the methods described herein, therapeutically effectiveamounts of the antibodies or antigen-binding fragments thereof,conjugates, and pharmaceutical compositions can be administered to asubject in need thereof, often for treating and/or preventing acondition or progression thereof. The antibodies or antigen-bindingfragments thereof, conjugates, or pharmaceutical compositions can affectthe physiology of the subject, such as the immune system, aninflammatory response, or other physiologic affect. A therapeuticallyeffective amount can vary depending on the severity of the disease, theage and relative health of the subject, the potency of the compoundsused, and other factors.

“Treatment,” “treat,” and/or “treating” refer to an intervention thatleads to any observable beneficial effect of the treatment or anyindicia of statistically significant success in the treatment oramelioration of the disease or condition, such as ameliorating a sign,symptom, or progression of a disease or pathological condition. Thebeneficial effect can be evidenced by, for example, a reduction, delayedonset, or alleviation of the severity of clinical symptoms of thedisease in a subject, a reduction in the frequency with which symptomsof a disease are experienced by a subject, a slower progression of thedisease, a reduction in the number of relapses of the disease, animprovement in the overall health or well-being of the subject, or byother parameters that are specific to the particular disease.

A prophylactic treatment meant to “prevent” a disease or condition(e.g., tumor formation or growth, in a subject or patient) is atreatment administered to a subject who does not exhibit signs of adisease or exhibits only early signs, for the purpose of decreasing therisk of developing pathology or further advancement of the earlydisease. For example, if an individual at risk of developing a tumor orother form of cancer is treated with the methods of the presentdisclosure and does not later develop the tumor or other form of cancer,then the disease has been prevented, at least over a period of time, inthat individual. A prophylactic treatment can mean preventingreccurrence of a disease or condition in a patient that has previouslybeen treated for the disease or condition, e.g., by preventing relapseor reccurance of cancer.

As used herein, the term “effective amount” or “effective dose” refersto a quantity of a specified antibody, conjugate, or composition thereofsufficient to achieve a desired (e.g., beneficial) effect in a subjectbeing treated with that compound, conjugate, or composition thereof,such as an amount sufficient to result in amelioration of one or moresymptoms of the disease being treated in a statistically significantmanner, delaying worsening of a progressive disease in a statisticallysignificant manner, or preventing onset of additional associatedsymptoms or diseases in a statistically significant manner, or anycombination thereof. In certain embodiments, an effective amount of anantibody, conjugate, or composition thereof is an amount sufficient toinhibit or treat the disease with minimal to no toxicity in the subject,excluding the presence of one or more adverse side effects. An effectiveamount or dose can be administered one or more times over a given periodof time. An effective amount or dose can depend on the purpose of thetreatment and can be ascertainable by one skilled in the art based on asubject's needs. When referring to an individual active ingredient,administered alone, an effective amount or dose refers to thatingredient alone. When referring to a combination, an effective amountor dose refers to combined amounts of the active ingredients that resultin the therapeutic effect, whether administered serially orsimultaneously.

Anti-Nectin-4 antibodies or antigen-binding fragments thereof,conjugates, and pharmaceutical compositions of this disclosure that canbe used in therapy can be formulated and dosages established in afashion consistent with good medical practice taking into account thedisease or condition to be treated, the condition of the individualpatient, the site of delivery of the composition, the method ofadministration and other factors known to practitioners.

Administration to a subject of an effective amount or dose of ananti-Nectin-4 antibody, conjugate, or composition thereof of thisdisclosure can be by one or more routes and can occur one or more timesover a given period of time. One of ordinary skill in the art wouldunderstand that the amount, duration and frequency of administration ofa compound, conjugate, or composition thereof of this disclosure to asubject in need thereof depends on several factors including, forexample, the health of the subject, the specific disease or condition ofthe patient, the grade or level of a specific disease or condition ofthe patient, the additional treatments the subject is receiving or hasreceived, or the like. Exemplary routes of administration includesubcutaneous, intravenous, intraarterial, subdural, intramuscular,intracranial, intrasternal, intratumoral, or intraperitoneal. In certainembodiments, the administration is subcutaneous. Additionally, acompound, conjugate, or composition thereof of this disclosure can beadministered to a subject by other routes of administration, forexample, by inhalation, or oral, dermal, intranasal or intrathecaladministration.

Anti-Nectin-4 antibodies or antigen-binding fragments thereof,conjugates, and compositions thereof the present disclosure can beadministered to a subject in need thereof in a first administration, andsubsequently in one or more additional administrations. The one or moreadditional administrations can be administered to the subject in needthereof minutes, hours, days, weeks or months following the firstadministration. Any one of the additional administrations can beadministered to the subject in need thereof less than 21 days, or lessthan 14 days, less than 10 days, less than 7 days, less than 4 days orless than 1 day after the first administration. The one or moreadministrations can occur more than once per day, more than once perweek or more than once per month. The administrations can be weekly,biweekly (every two weeks), every three weeks, monthly or bimonthly.

In some aspects, the present disclosure provides a method of treatingcancer. In certain embodiments, the present disclosure provides a methodof treating cancer comprising administering to a subject in need thereofan effective amount of: (a) an anti-Nectin-4 antibody conjugatecomprising a compound of Formula (X-1)-(X-9), Category A of Formula(IA), (IB), (IIA), (IIB), (IIC), (IIIA), (IIIB), (IVA), (IVB), or (IVC),or pharmaceutically acceptable isomer, racemate, hydrate, solvate,isotope, or salt thereof; or (b) a composition of (a).

In any of the embodiments herein, an antibody conjugate composition foruse in the methods of treating disease (such as cancer) has an averageDAR of the conjugate ranging from about 2 to about 8, about 1 to about3, or about 3 to about 5. In other embodiments, an antibody conjugate ina composition will have an average DAR ranging from 1 to about 10, from1 to about 9, from 1 to about 8, from 1 to about 6, from 1 to about 3,from about 2 to about 8, from about 2 to about 6, from about 2.5 toabout 5.5, from about 2.5 to about 4.5, from about 2 to about 4, fromabout 3.5 to about 5.5, from about 3 to about 5, from about 3.5 to about4.5, or from about 3 to about 4. In certain embodiments, the average DARfor the conjugates of a composition used in the methods of treatmentwill be about 1.5, about 2, about 2.5, about 3, about 3.5, about 4,about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5,or about 8. In certain embodiments, an anti-Nectin-4 antibody conjugateof this disclosure comprises from 1-20 myeloid agonist compounds (e.g.,TLR8 agonists) per antibody, preferably ranges from 1 to about 8, about3 about 5, or 1 to about 3 myeloid agonist compounds (e.g., TLR8agonists) per antibody.

In various embodiments, methods of treating a cancer (e.g., bladder,breast, lung, cervical, HPV+ cancer or tumor) are provided, comprisingadministering an effective amount of an antibody or an antigen-bindingfragment thereof, a conjugate, or a pharmaceutical composition providedherein to a subject in need thereof. In some embodiments, the cancer isselected from bladder cancer, breast cancer, lung cancer, head and neckcancer, cervical cancer, pancreatic cancer, gastric cancer, esophagealcancer, and uterine cancer. In some embodiments, the bladder cancer isurothelial cancer. In some embodiments, the breast cancer is triplenegative breast cancer. In some embodiments, the lung cancer isnon-small cell lung cancer (NSCLC). In some embodiments, the NSCLC issquamous cell carcinoma or lung adenocarcinoma. In some embodiments, thehead and neck cancer is head and neck squamous cell carcinoma (HNSCC).In some embodiments, the cervical cancer is cervical squamous cellcarcinoma or endocervical adenocarcinoma. In some embodiments, thepancreatic cancer is pancreatic adenocarcinoma. In some embodiments, theesophageal cancer is esophageal adenocarcinoma. In some embodiments, theuterine cancer is uterine corpus endometrial carcinoma.

In various embodiments, methods of treating a Nectin-4-expressing cancerare provided, comprising administering an effective amount of anantibody or an antigen-binding fragment thereof, a conjugate, or apharmaceutical composition provided herein to a subject in need thereof.In some embodiments, the cancer is selected from bladder cancer, breastcancer, lung cancer, head and neck cancer, cervical cancer, pancreaticcancer, esophageal cancer, and uterine cancer. In some embodiments, thebladder cancer is urothelial cancer. In some embodiments, the breastcancer is triple negative breast cancer. In some embodiments, the lungcancer is non-small cell lung cancer (NSCLC). In some embodiments, theNSCLC is squamous cell carcinoma or lung adenocarcinoma. In someembodiments, the cervical cancer is cervical squamous cell carcinoma orendocervical adenocarcinoma. In some embodiments, the pancreatic canceris pancreatic adenocarcinoma. In some embodiments, the esophageal canceris esophageal adenocarcinoma. In some embodiments, the uterine cancer isuterine corpus endometrial carcinoma.

In some embodiments, the methods of treating cancer or treating aNectin-4-expressing cancer further comprise administering an additionaltherapy to the subject, such as, for example, small molecule inhibitors(including small molecule antagonists), CAR-T cells, therapeuticantibodies, antibody-drug conjugates, chemotherapeutic agents, ionizingradiation, and other anti-cancer drugs.

Examples of therapeutic antibodies contemplated as further therapeuticagents include anti-TIGIT antibodies, such as tiragolumab, vibostolimab,domvanalimab, BMS-986207, ASP8374, and BGB-A1217. In certainembodiments, an anti-TIGIT antibody may be combined with an additionalantibody (e.g., an anti-PD-1 or anti-PD-L1 antibody) to be used with ananti-Nectin-4 antibody or an anti-Nectin-4 antibody conjugated to amyeloid cell agonist (such as a TLR8 agonist) as provided in thisdisclosure. The combination therapy using an anti-Nectin-4 antibody oranti-Nectin-4 antibody conjugated to a myeloid cell agonist provided inthis disclosure, an anti-TIGIT antibody, and optionally an anti-PD-1 oranti-PD-L1 antibody may be used in treating various types of cancer,including lung cancer (e.g., small lung cell cancer and non-small celllung cancer), gastro-esophageal junction cancer, and esophageal cancer.

Examples of small molecule antagonists contemplated as furthertherapeutic agents include adenosine receptor antagonist, such as AB928(Arcus), CPI-444 (Corvus), PBF-509 (Pablobio), MK-3814 (Merck), andAZD4635 (AstraZeneca). In certain embodiments, an adenosine receptorantagonist may be combined with an additional antibody (e.g., ananti-PD-1 or anti-PD-L1 antibody) to be used with an anti-Nectin-4antibody or an anti-Nectin-4 antibody conjugated to a myeloid cellagonist as provided in this disclosure.

Examples of chemotherapeutic agents contemplated as further therapeuticagents include alkylating agents, such as nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide (IFEX®), melphalan(Alkeran®), and chlorambucil); bifunctional chemotherapeutics (e.g.,bendamustine); nitrosoureas (e.g., carmustine (BCNU, BiCNU®;polifeprosan 20 implant (Gliadel®)), lomustine (CCNU), and semustine(methyl-CCNU)); ethyleneimines and methyl-melamines (e.g.,triethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), andhexamethylmelamine (HMM, altretamine)); alkyl sulfonates (e.g., busulfan(Myleran®), busulfan injection (Busulfex®)); and triazines (e.g.,dacabazine (DTIC)); antimetabolites, such as folic acid analogues (e.g.,methotrexate (Folex®), trimetrexate, and pemetrexed (multi-targetedantifolate)) and capecitabine (Xeloda®); pyrimidine analogues (such as5-fluorouracil (5-FU, Adrucil®, Efudex®), fluorodeoxyuridine,tezacitabine, gemcitabine, cytosine arabinoside (AraC, cytarabine(Cytosar-U®); cytarabine liposome injection (DepoCyt®)), 5-azacytidine,and 2,2′-difluorodeoxycytidine); purine analogues (e.g.,6-mercaptopurine (Purinethol®), 6-thioguanine, azathioprine,2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA),fludarabine phosphate (Fludara®), 2 chlorodeoxyadenosine (cladribine,2-CdA)); Type I topoisomerase inhibitors such as camptothecin (CPT),topotecan (Hycamptin®), and irinotecan (Camptosar®); natural products,such as epipodophylotoxins (e.g., etoposide (Vepesid®) and teniposide(Vumon®)); vinca alkaloids (e.g., vinblastine (Velban®), vincristine(Oncovin®), and vinorelbine (Navelbine®)); anti-tumor antibiotics suchas actinomycin D (dactinomycin, Cosmegan®), doxorubicin hydrochloride(Adriamycin®, Rubex®), mitoxantrone (Novantrone®), and bleomycin sulfate(Blenoxane®); radiosensitizers such as 5-bromodeozyuridine,5-iododeoxyuridine, and bromodeoxycytidine; platinum coordinationcomplexes such as cisplatin (Platinol®), carboplatin (Paraplatin®), andoxaliplatin (Eloxatin®); substituted ureas, such as hydroxyurea(Hydrea®); microtubule inhibitors such as paclitaxel (Taxol®) anddocetaxel (Taxotere®); immunosuppressive agents such as cyclophosphamide(Cytoxan® or Neosar®); hormone-based compound such as anastrozole(Arimidex®), exemestane (Aromasin®), letrozole (Femara®), fulvestrant(Faslodex®), and bicalutamide (Casodex®) and tamoxifen citrate(Nolvadex®); an anti-inflammatory agent such as dexamethasone; ananti-androgen compound such as flutamide (Eulexin®); an anthracyclinecompound such as idarubicin (Idamycin®, Zavedos®) and epirubicin(Ellence®); bioreductive anti-cancer agent such as tirapazamine(Tirazone®); serine/threonine kinase inhibitors such as CDK4/6inhibitors abemaciclib (Verzenio®), palbociclib (Ibrance®), andribociclib (Kisqali®); and methylhydrazine derivatives such as Nmethylhydrazine (MIH) and procarbazine.

In some embodiments, wherein the disease is urothelial cancer, theadditional therapeutic agent is selected from dose-dense methotrexate,vinblastine, doxorubicin, and cisplatin (ddMVAC), gemcitabine/cisplatin,gemcitabine/carboplatin, atezolizumab, pembrolizumab, nivolumab,durvalumab, avelumab, erdafitinib, and enfortumab vedotin.

In some embodiments, wherein the disease is triple negative breastcancer, the additional therapeutic agent is selected from a taxane(e.g., paclitaxel, docetaxel), anthracycline, capecitabine, gemcitabine,eribulin, a PD1/PDL1 inhibitor (e.g., nivolumab, pembrolizumab,atezolizumab), antibody-drug conjugate (ADC) (e.g., sacituzumabgovitecan, ladiratuzumab vedotin), a PARP inhibitor, PTEN/AKT/PI3Kinhibitor, MEK inhibitor, or any combination thereof.

In some embodiments, wherein the disease is NSCLC (squamous cellcarcinoma or lung adenocarcinoma), the additional therapeutic agent isselected from a platinum-based therapeutic (e.g., carboplatinum), ataxane (e.g., paclitaxel docetaxel), radiation, a PD1/PDL1 inhibitor(e.g., nivolumab, pembrolizumab, atezolizumab), a kinase inhibitor (suchas an EGFR, ALK, BRAF, MEK, NTRK, or VEGFR inhibitor; e.g., osimentinib,crizotinib, dabrafenib, tramentinib, erectinib, nintedanib), otherantibody (such as anti-EGFR, anti-VEGF/anti-VEGFR; e.g., erbitux,avastin, ramucirumab), or any combination thereof.

In some embodiments, wherein the disease is pancreatic adenocarcinoma,the additional therapeutic agent is selected from radiation, aplatinum-based therapy (e.g., oxaliplatin), an anti-metabolite (e.g.,capecitabine, gemcitabine, irinotecan), a taxane (e.g., NAB-paclitaxel,paclitaxel, docetaxel), a kinase inhibitor (e.g., erlotinib), a PARPinhibitor (e.g., olaparib), a metabolic therapy (e.g., 5-fluorouracil(plus leucovorin), devimistat), a checkpoint inhibitor (e.g., anti-PD1,anti-PDL-1, anti-CTLA4), an oncolytic virus (e.g., pelareorep), or anycombination thereof.

In some embodiments, wherein the disease is head and neck cancer, theadditional therapeutic agent is selected from radiation, aplatinum-based therapy (e.g., cisplatin, carboplatin), taxanes (e.g.,paclitaxel, docetaxel), a metabolic therapy (e.g., 5-fluorouracil), ananti-EGFR antibody (e.g., cetuximab); a PD1/PDL-1 inhibitor (e.g.,pembrolizumab, nivolumab, envafolimab), a tyrosine kinase inhibitor(e.g., erlotinib, lapatinib), an oncolytic virus (e.g., talimogenelaherparepvec); a CAR T cell therapy (e.g., KITE HPV-16, KITE HPV-17),or any combination thereof.

In some embodiments, the additional therapeutic agent is an anti-TROP2antibody-drug conjugate (e.g., sacituzumab govitecan, DS-1062, SKB264,BAT8003). In certain embodiments, such combinations of an anti-Nectin-4antibody conjugated to a myeloid cell agonist with an anti-TROP2antibody-drug conjugate are used to treat certain cancers, such asbladder cancer, HNSCC, or TNBC.

In some embodiments, wherein the disease is a HPV+ tumor (e.g., HNSCCand cervical cancer), the additional therapeutic agent is a CAR T celltherapy (e.g., KITE-439).

In some embodiments, wherein the disease is gastric cancer, NSCLC, orbladder cancer, the additional therapeutic agent is a PD-L1 smallmolecule inhibitor (e.g., GS-4224, aurigenel, BMSpep-57, BMS-103,BMS-142, BMS-1166).

In various embodiments, the method comprises administering an effectiveregimen that results in a T max of the conjugate of greater than 4 hoursfollowing each administration of the conjugate. In some embodiments, theeffective regimen results in a T max greater than 6 hours, greater than8 hours, greater than 10 hours, greater than 12 hours, or greater than15 hours following each administration of the conjugate.

In certain embodiments, the methods include administration of animmune-stimulatory conjugate, or a pharmaceutical composition thereof,to a subject in need thereof in an effective regimen to activate,stimulate or augment an immune response against a disease treatable witha TLR agonist (e.g., cancer). The antibody or antigen binding fragmentthereof of the conjugate recognizes an antigen associated with thedisease or disease state, such as Nectin-4.

In certain embodiments, the methods include administration of acombination therapy comprising a conjugate provided herein to a subjectin need thereof to activate, stimulate or augment an immune responseagainst tumor cells of a solid tumor, such as bladder cancer, breastcancer, lung cancer, head and neck cancer, cervical cancer, pancreaticcancer, esophageal cancer, and uterine cancer. In some such embodiments,the antibody or antigen binding fragment thereof of the conjugaterecognizes an antigen on the tumor cells, e.g., Nectin-4.

In some cases, treatment comprises reduced tumor growth. In some cases,treatment comprises tumor arrest.

One of ordinary skill in the art would understand that the amount,duration and frequency of administration of a conjugate or combinationtherapy described herein to a subject in need thereof depends on severalfactors including, for example but not limited to, the health of thesubject, the specific disease or condition of the subject, the grade orlevel of a specific disease or condition of the subject, the additionaltherapeutics the subject is being or has been administered, and thelike.

In some aspects of practicing the methods described herein, theconjugates are administered in an effective regimen of at least two orat least three cycles. Each cycle can optionally include a resting stagebetween cycles. Cycles of administration can be of any suitable length.In some embodiments, each cycle is a week (7 days), 10 days, every twoweeks (14 days or biweekly), every three week (21 days) or every fourweeks (28 days). In some embodiments, each cycle is a month. In someembodiments, at least two doses of the immune-stimulatory conjugate areadministered more than 7 days apart, or more than 10 days apart. In someembodiments, at least one dose of the conjugate is administered morethan 7 days, or more than 10 days, after the initial dose of theconjugate.

In certain embodiments, the total dose of the conjugate within atreatment cycle or the dose of the conjugate per administration is fromabout 0.1 to about 10 mg/kg. In some embodiments, the total dose withina treatment cycle or the dose of the conjugate per administration isfrom about 0.5 to about 7.5 mg/kg. In some embodiments, the total dosewithin a treatment cycle or the dose of the conjugate per administrationis from about 0.5 to about 5 mg/kg. In some embodiments, the total doseof the conjugate within a treatment cycle or the dose of the conjugateper administration is from about 0.5 to about 4 mg/kg. In someembodiments, the total dose of the conjugate within a treatment cycle orthe dose of the conjugate per administration is from about 0.5 to about3.5 mg/kg. In some embodiments, the total dose of the conjugate within atreatment cycle or the dose of the conjugate per administration is fromabout 0.5 to about 2 mg/kg.

In certain embodiments, the total dose of the conjugate within atreatment cycle is from about 0.1 to about 100 mg/kg, such as about 0.1to about 50 mg/kg, about 0.1 to about 25 mg/kg, about 0.1 to about 20mg/kg, about 0.1 to about 15 mg/kg, about 0.1 to about 10 mg/kg, about0.5 to about 100 mg/kg, about 0.5 to about 50 mg/kg, about 0.5 to about25 mg/kg, about 0.5 to about 20 mg/kg, about 0.5 to about 15 mg/kg,about 0.5 to about 10 mg/kg, about 1 to about 100 mg/kg, about 1 toabout 50 mg/kg, about 1 to about 25 mg/kg, about 1 to about 20 mg/kg,about 1 to about 15 mg/kg, or about 1 to about 10 mg/kg.

In certain embodiments, the dose of the conjugate per administration isfrom about 0.1 to about 100 mg/kg, such as about 0.1 to about 50 mg/kg,about 0.1 to about 25 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 toabout 15 mg/kg, about 0.1 to about 10 mg/kg, about 0.5 to about 100mg/kg, about 0.5 to about 50 mg/kg, about 0.5 to about 25 mg/kg, about0.5 to about 20 mg/kg, about 0.5 to about 15 mg/kg, about 0.5 to about10 mg/kg, about 1 to about 100 mg/kg, about 1 to about 50 mg/kg, about 1to about 25 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg,or about 1 to about 10 mg/kg.

In certain preferred embodiments, the total dose of the conjugate withina treatment cycle is about 0.1 to about 25 mg/kg. In certain otherpreferred embodiments, the total dose of the conjugate within atreatment cycle is about 0.5 to about 20 mg/kg. In certain preferredembodiments, the total dose of the conjugate within a treatment cycleabout 0.1, about 0.5, about 2, about 4, about 6, about 8, about 10,about 12, about 14, or about 16 mg/kg.

In certain preferred embodiments, the dose of the conjugate peradministration is about 0.1 to about 25 mg/kg. In certain otherpreferred embodiments, the dose of the conjugate per administration isabout 0.5 to about 20 mg/kg. In certain preferred embodiments, the doseof the conjugate per administration about 0.1, about 0.5, about 2, about4, about 6, about 8, about 10, about 12, about 14, or about 16 mg/kg.

In certain embodiments, the conjugate is administered subcutaneously,and the total dose of the conjugate within a treatment cycle is fromabout 0.1 to about 100 mg/kg, such as about 0.1 to about 50 mg/kg, about0.1 to about 25 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 to about15 mg/kg, about 0.1 to about 10 mg/kg, about 0.5 to about 100 mg/kg,about 0.5 to about 50 mg/kg, about 0.5 to about 25 mg/kg, about 0.5 toabout 20 mg/kg, about 0.5 to about 15 mg/kg, about 0.5 to about 10mg/kg, about 1 to about 100 mg/kg, about 1 to about 50 mg/kg, about 1 toabout 25 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, orabout 1 to about 10 mg/kg.

In certain embodiments, the conjugate is administered subcutaneously,and the dose of the conjugate per administration is from about 0.1 toabout 100 mg/kg, such as about 0.1 to about 50 mg/kg, about 0.1 to about25 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 to about 15 mg/kg,about 0.1 to about 10 mg/kg, about 0.5 to about 100 mg/kg, about 0.5 toabout 50 mg/kg, about 0.5 to about 25 mg/kg, about 0.5 to about 20mg/kg, about 0.5 to about 15 mg/kg, about 0.5 to about 10 mg/kg, about 1to about 100 mg/kg, about 1 to about 50 mg/kg, about 1 to about 25mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, or about 1to about 10 mg/kg.

In certain embodiments, the conjugate is administered subcutaneously,and the total dose of the conjugate within a treatment cycle is about0.1 to about 25 mg/kg. In certain other preferred embodiments, theconjugate is administered subcutaneously, and the total dose of theconjugate within a treatment cycle is about 0.5 to about 20 mg/kg. Incertain preferred embodiments, the conjugate is administeredsubcutaneously, and the total dose of the conjugate within a treatmentcycle about 0.1, about 0.5, about 2, about 4, about 6, about 8, about10, about 12, about 14, or about 16 mg/kg.

In certain embodiments, the conjugate is administered subcutaneously,and the dose of the conjugate per administration is about 0.1 to about25 mg/kg. In certain other preferred embodiments, the conjugate isadministered subcutaneously, and the dose of the conjugate peradministration is about 0.5 to about 20 mg/kg. In certain preferredembodiments, the conjugate is administered subcutaneously, and the doseof the conjugate per administration about 0.1, about 0.5, about 2, about4, about 6, about 8, about 10, about 12, about 14, or about 16 mg/kg.

Application of immune-stimulatory conjugates described herein showssubstantial benefit in directing a subject's own immune response tocells of a particular site of disease or disorder, such as cellsassociated with the disease or disorder. Activating or stimulating animmune response directed to targeted cells facilitates the reduction,inhibition of proliferation, inhibition of growth, inhibition ofprogression, inhibition of metastasis or otherwise inhibition up to andincluding in some cases clearance of the targeted cells. Thus, in somecases a targeted immune response activation or stimulation leads toinhibition of disease progression, or alleviation of at least onesymptom of a manifest disease in a patient, up to and in some casesincluding complete elimination of from one symptom to an entire diseasestate in a subject.

In particular, the methods disclosed herein are well suited for use withimmune stimulatory conjugates, such as immune stimulatory conjugatesthat direct an immune response in a subject to a particular disorder ordisease location, cell type or cell. Accordingly, practice of somemethods herein comprises selection of a suitable subject such as asubject to be subjected to or undergoing a treatment with a conjugatethat directs a benzazepine or benzazepine-like compound of the conjugateto a particular disorder or disease site, cell type or cell. Often, thesubject is selected for practice of the method due to having at leastone symptom of a disease or disorder, or projected to develop at leastone symptom of a disease or disorder (such as a subject in remission andat risk for relapse), suitable for treatment by a conjugate as disclosedherein. Some diseases are selected not based upon or not based solely ondisease type, but upon detection or presence of a suitable epitope on atumor, cell type or particular cell that facilitates localization of animmune-stimulatory conjugate to the epitope.

EXAMPLES Example 1 Generation and Humanization of Anti-Nectin-4Monoclonal Antibodies

Hybridomas producing monoclonal antibodies (mAbs) specific for humanNectin-4 were prepared from Balb/C and NZB/NZW mice were immunized withNectin-4-mouse Fc (IgG2a) fusion protein and Nectin-4 transfected 3T3cells using standard procedures. Antibody heavy and light chainsequences were obtained, amplified, and cloned. Clone supernatantscontaining the expressed mAbs were screened for the certain criteria,including, for example, high titer, binding to human Nectin-4 protein,binding to cells expressing Nectin-4, and cross-reactivity withCynomolgus macaque cells. Based on the initial selection criteria, onemAb (D6C) was expressed and isolated.

For humanization of the D6C VH region (SEQ ID NO:9), the 3 CDR loops asdefined by Kabat were grafted into the human germline sequence VH3-07with JH6 to generate hzD6C VH (SEQ ID NO: 10).

For humanization of the D6C VL region (SEQ ID NO:11), the 3 CDR loops asdefined by Kabat were grafted into the human germline sequence VKII-O1with JK4 to generate hzD6C VL (SEQ ID NO:12). In addition, severalvariants of hzD6C VLv1 were constructed to contain one or more mouseback mutations in framework region 1 (FR1), CDR1, or both. Variant 1 hasa FR1 mutation (hzD6.1C VL 12V, SEQ ID NO: 13), and variants 2 and 3each contain a different CDR1 mutation to remove a potential deamidationsite (hzD6.2C VL G34A (position 30d under Kabat), SEQ ID NO: 14; andhzD6.3C VL N33Q (position 30c under Kabat), SEQ ID NO: 15;respectively). Variants 4 and 5 each have a FR1 and a CDR1 mutation(hzD6.4C VL I2V N33Q (positions 2 and 30c under Kabat), SEQ ID NO: 16;and hzD6.5C VL I2V G34A (positions 2 and 30d under Kabat), SEQ ID NO:17; respectively).

The humanized D6C VH region was combined with human IgG1 (SEQ ID NO: 18)heavy chain constant region, and each humanized D6C VL region wascombined individually with the human kappa light chain constant region(SEQ ID NO:20). The humanized D6C heavy chain (SEQ ID NO:24) wasco-transfected with each humanized light chain (SEQ ID NOS:26-31)individually into CHO cells in a 30 mL culture, using the parentalchimeric antibody, D6C, as a benchmark. A total of 7 mAbs weregenerated, purified, and analyzed for titer, purity, and binding.

The highest titers were observed for hzD6.1C VL-hzD6.4C VL (481, 432,476, and 426 mg/L, respectively), whereas the titers for chimeric D6CVL, hzD6C VL, and hzD6.5C VL were substantially lower (206, 345, and 177mg/L, respectively). Analytical size-exclusion chromatography was usedto measure the percent purity (% protein of interest, POI), which ispreferably 95% or greater. There was a reduction in percent purity seenwith the chimeric D6C (84%) and the CDR grafted hzD6C VL (94%). The I2V,N33Q, and G34A mutations in hzD6.1C VL (98%), hzD6.2C VL (95%), andhzD6.3C VL (96%), respectively, as well as the IV2 N33Q double mutationhzD6.4C VL (99%), resolve the purity issue. In contrast, the IV2 G34Adouble mutation in hzD6.5C VL significantly reduces the purity (75%).Finally, for binding, the hzD6C VL, hzD6.1C VL, hzD6.2C VL, and hzD6.5CVL showed the best binding (KD=17, 21, 32, and 44, respectively),whereas the hzD6.3C VL and hzD6.4C VL constructs showed reduces bindingto human Nectin-4 (KD=286 and 204, respectively) and no binding to mouseNectin-4. Antibody epitope binning experiments utilizing Octet®(ForteBio, Inc.) kinetic analysis revealed a competitive blockingprofile similar to humanized mAb Ha2-22 (a known anti-Nectin-4 mAb; see,e.g., U.S. Pat. Publication No. 2012/0078028)

Based on these results, mAbs containing the D6C VH region and hzD6.1CVL, and containing the D6C VH region and hzD6.2C VL, were selected forfurther use and analysis.

Example 2 Anti-Nectin-4 Immunoconjugates Bind to Nectin-4 ExpressingCell Lines

To examine the ability of anti-Nectin-4-TLR8 agonist conjugates to bindto Nectin-4-expressing cell lines (HEK-293 cells transfected with humanor cynomolgus Nectin-4, or Nectin-4 expressing tumor cell lineMDA-MB-175-VII), cells were plated at about 5×10⁴ cells/well andcontacted with titrating concentrations of unconjugated anti-Nectin-4antibodies (hzD6.2C, hzD6.1C, D6C and anti-Nectin-4 mAb IgG1 (ahumanized mAb having CDRs from Ha22-2), anti-Nectin-4-TLR8 agonistimmunoconjugates (hzD6.2C-Compound 2.14, hzD6.1C-Compound 2.14, D6CIgG1-Compound 2.14, and anti-Nectin-4 IgG1-Compound 2.14), or antibodyisotype control (Digoxin IgG1) in FACS Wash (FW-PBS, 2% FBS, 1 mM EDTA)for 30 mins, at 4° C. followed by secondary anti-huIgG1-PE staining inFW for 30 mins, at 4° C. After incubations, cells were washed with FWand then analyzed on a flow cytometer.

FIGS. 1A-1C show that the humanized anti-Nectin-4 immunoconjugates(hzD6.2C-Compound 2.14, hzD6.1C-Compound 2.14, and anti-Nectin-4IgG1-Compound 2.14), as well as the parental antibody TLR8 agonistconjugate (D6C IgG1-Compound 2.14), all bind to the human and thecynomolgus Nectin-4-expressing cells with a similar EC₅₀ as theirunconjugated anti-Nectin-4 antibody counterparts (hzD6.2C, hzD6.1C, D6Cand Nectin4 mAb IgG1). Thus, the conjugation of the TLR8 agonist to theantibodies does not affect their ability to specifically bind toNectin-4. In addition, Nectin-4 binding by the hzD6.2C-Compound 2.14,which has a mutation in CDR1, was surprisingly not affected.

Example 3 Human PBMC TNF-Alpha Production Induced by Anti-Nectin-4 TLR8Agonist Conjugates in the Presence of Nectin-4 Expressing Tumor CellLines

Production of TNF-α from peripheral blood mononuclear cells (PBMCs)co-cultured with Nectin-4 expressing tumor cell lines when contactedwith anti-Nectin-4-TLR8 agonist conjugates was examined. Briefly, PBMCswere isolated from normal human donor peripheral blood using SepMate™-50PBMC Isolation Tubes (STEMCELL Technologies) according to manufacturer'sinstructions. Isolated PBMCs were cultured with the Nectin-4 expressingtumor cell line MDA-MB-175-VII (ATCC) or the Nectin-4 negative cell lineHEK-293 (ATCC) at a 5:1 ratio in the presence of titrated concentrationsof anti-Nectin-4-TLR8 agonist antibody conjugates, unconjugatedanti-Nectin-4 antibody controls, or an isotype control-TLR8 agonistconjugate. After 24 hours, the cell-free supernatants were collected andstored at −80° C. prior to analysis. TNF-α levels in the cell-freesupernatants were quantified using the TNF-α (human) AlphaLISA®Detection Kit (Perkin Elmer) according to manufacturer's instructions.

FIGS. 2A-2B show that anti-Nectin-4-TLR8 agonist conjugates inducedTNF-α production in a dose-dependent manner from human PBMCs in thepresence of the Nectin-4 expressing MDA-MB-175-VII tumor cell line (FIG.2A), but not in the presence of HEK-293 cells lacking expression ofNectin-4 (FIG. 2B). Unexpectedly, the humanized derivativeshzD6.1C-Compound 2.14 and hzD6.2C-Compound 2.14 not only showed twicethe potency in inducing TNF-α production (EC₅₀ of 0.33 nM and 0.37 nM,respectively [average of three experiments]) compared to the parentalantibody TLR8 agonist conjugate (D6C IgG1-Compound 2.14; EC₅₀ of 0.83nM), but also induced about a 3-fold increase in maximal TNF-αproduction (see FIG. 2A). Moreover, while the humanized anti-Nectin-4IgG1-Compound 2.14 conjugate (Ha22-2 anti-Nectin-4 antibody thatcross-blocks the D6C antibodies of this disclosure) demonstrated anincreased potency (EC₅₀ of 0.13 nM), the humanized derivativeshzD6.1C-Compound 2.14 and hzD6.2C-Compound 2.14 of this disclosure stillunexpectedly induced about a 3-fold increase in maximal TNF-α production(see FIG. 2A). TNF-α production by PBMCs was not induced in the presenceof the Nectin-4 expressing tumor cell line with unconjugated antibodies,which indicates that TLR8 agonism is needed for TNF-α release.Furthermore, none of the conjugated or unconjugated antibodiesstimulated TNF-α production from PBMCs in the absence ofNectin-4-expressing tumor cells indicating that the activity isdependent upon Nectin-4 expression. Low level PBMC TNF-α production wasobserved with the Nectin-4 negative HEK-293 cell line only in culturescontaining the highest concentrations of anti-Nectin-4-TLR8 conjugatestested. Similar results were observed with cynomolgus PBMC cultures from4 separate donors (data not shown).

Example 4 Anti-Nectin-4-TLR8 Agonist Surrogate Immunoconjugates InduceTNFalpha Production in Macrophages

Rodents do not express a functional homolog of TLR8 and attempts bymultiple groups to generate relevant human TLR8 transgenic mice havebeen unsuccessful (Wang, J. Biol. Chem. 257:37427, 2006; Guiducci, J.Exp. Med 270:2903, 2013). Like TLR8 in humans, and in contrast to TLR7in humans, TLR7 in mice is expressed in myeloid cells, like macrophages.Thus, for murine experiments, an anti-Nectin-4-TLR7 agonist conjugate isused as a surrogate for human anti-Nectin-4-TLR8 agonist conjugates). Toexamine whether the anti-Nectin-4-TLR7 agonist conjugate surrogate couldinduce TNFα expression murine bone marrow-derived macrophages (BMDM),such cells were obtained by harvesting bone marrow cells from a Balb/cmice and differentiating in vitro into macrophages for 7 days in mediasupplemented with murine M-CSF.

After differentiation, murine BMDM were harvested and counted beforebeing seeded at about 8.0×10⁴ cells/well in 96-well flat bottommicrotiter plates in assay media (RPMI, 10% Fetal Bovine Serum, 1 mMSodium Pyruvate, IX GlutaMAX-1, 1× Non-Essential Amino Acids, 10 mMHEPES, 50 units/mL Penicillin and 50 ug/mL Streptomycin).Nectin-4-expressing HEK-293 cells were then added (about 4.0×10⁴cells/well) along with titrating concentrations of unconjugatedanti-Nectin-4 antibodies (D6C mAb IgG2a, and anti-Nectin-4 mAb mIgG2a),anti-Nectin-4-TLR7 agonist immunoconjugates (D6C mIgG2a-Compound 4.1 andanti-Nectin-4 mIgG2a-Compound 4.1), or control TLR7 agonistimmunoconjugate (Isotype Control-Compound 4.1). Mock transfected HEK-293cells were used as a negative control. After 24 hours culture,supernatants were collected and murine TNFα levels in the supernatantswere determined by ELISA.

The surrogate anti-Nectin-4-TLR7 agonist immunoconjugates (D6CmIgG2a-Compound 4.1 and anti-Nectin4 mIgG2a-Compound 4.1) were activewhen crosslinked with HEK-293 cells transfected with Nectin-4,stimulating production of TNFα from murine BMDM in a dose dependentmanner (FIG. 3A) similar to the anti-Nectin4-TLR8 agonistimmunoconjugates in the human PBMC assay system. In contrast, theunconjugated anti-Nectin-4 antibodies (D6C mAb IgG2a, and anti-Nectin-4mAb mIgG2a) stimulated low levels of TNFα production from murine BMDM inthe presence of HEK-293 cells transfected with Nectin-4. Furthermore,none of the immunoconjugates or unconjugated antibodies stimulated TNFαproduction from murine BMDM in the presence of HEK-293 cells lackingexpression of Nectin-4 (FIG. 3B).

Example 5 In Vivo Activity of Nectin4-TLR7 Agonist Conjugate Surrogatein Mice Bearing Human Nectin-4 Expressing EMT6 Tumors

To examine the in vivo efficacy of the anti-Nectin-4-TLR7 agonistconjugate surrogate (D6C mIgG2a-Compound 4.1), 7-9 week old femaleBalb/c mice (The Jackson Laboratory) were orthotopically implantedsubcutaneously into the mammary fat pad with about 1×10⁵ humanNectin-4-expressing EMT6 syngeneic breast cancer cells. Tumor volumeswere measured using the equation: VOL=(L×W²)/2. Once tumors reachedapproximately 150 mm³ (Day 0), mice were sorted into groups of 10 andeach group was individually dosed subcutaneously with 10 mg/kg of oneof: (a) mouse IgG2a (mIgG2a) isotype control, (b) D6C mIgG2a(unconjugated), or (c) D6C mIgG2a-Compound 4.1, every 7 days for a totalof 3 doses. The isotype control antibody was an anti-digoxin antibody.Tumor volumes were recorded 3 times per week for at least 28 days.Statistical analysis was performed using Log-rank (Mantel-Cox) test.

FIG. 4 shows that the mice treated with the surrogate of theanti-Nectin-4-TLR8 agonist conjugate resulted in delayed tumor growthand a survival advantage as compared to those treated with theunconjugated anti-Nectin-4 antibody or the isotype control.

Example 6 Intra-Tumoral Chemokines and Cytokines in Tumor-Bearing MiceTreated with a Surrogate of a Nectin4-TLR8 Conjugate

To examine the effect of treating tumor-bearing mice with Nectin4-TLR7conjugate (D6C mIgG2a-Compound 4.1), a surrogate for human Nectin4-TLR8conjugate, chemokine and cytokine production was evaluated in thetumors. Briefly, female BALB/c mice (Jackson Laboratory) were inoculatedin the mammary fat with 1×10⁵ human Nectin4-expressing EMT6 syngeneicbreast cancer cells. Once tumors reached a volume of approximately 100mm³ (Day 0), mice were sorted into groups of 6 mice and each group wasindividually dosed once subcutaneously with 10 mg/kg of one of: (a) D6CmIgG2a (unconjugated control) or (b) D6C mIgG2a-Compound 4.1. Two dayslater, tumors were excised and weighed. In a separate study, mice wereinoculated, treated as described above and tumors were excised at day 5.Tumors were placed in 500 mL RPMI (Gibco) and mechanically dissociatedon ice. The resulting supernatants were analyzed by Luminex (Millipore)for intra-tumoral levels of chemokines MCP-1 and IP 10 (indicators ofmyeloid cell activation) and cytokines IFN-γ and IL-2 (indicators of Tcell and/or NK cell activation). Data is expressed as picogram ofanalyte per gram of starting tissue. Statistical analysis was performedusing Log-rank (Mantel-Cox) test.

As shown in FIG. 5A, MCP-1 and IP 10 production was significantlyincreased by treatment with D6C mIgG2a-Compound 4.1 compared to D6CmIgG2a at day 2, indicating potent myeloid cell activation. Moreover,IFN-γ and IL-2 production was augmented at day 5 in tumors of micetreated with D6C mIgG2a-Compound 4.1 (FIG. 5B), which indicates that Tcells and NK cells are being activated. Overall, these data indicatethat treatment with D6C mIgG2a-Compound 4.1 conjugate can promote anenhanced innate immune response driven by myeloid cell activation, whichin turn is capable of nucleating an adaptive immune response byindirectly activating T and NK cells within the tumor.

Example 7 Specificity of Anti-Nectin-4 Monoclonal Antibodies forNectin-4

Analysis of hzD6.2C monoclonal antibody (mAb) for binding to humanNectin-1, Nectin-2, Nectin-3, and Nectin-4 extracellular domains (ECDs)was performed using Octet® Red 96 instrument (ForteBio, Inc.).Biotinylated hzD6.2C mAb was immobilized on streptavidin biosensors andincubated with hFc-tagged human Nectin-1, -2, -3, and -4 ECD dimericproteins (Sino Biological) at varying concentrations ranging from 1.2 nMto 300 nM in PBS/1% BSA/0.2% Tween 20. The experimental measurementswere performed as follows: (1) baseline acquisition (30 s); (2)biotinylated hzD6.2C mAb loading onto streptavidin biosensor (120 s);(3) second baseline acquisition (30 s); (4) association of interactingdimeric Nectin 1/2/3/4 ECD proteins for k_(on) measurement (90 s); and(5) dissociation of interacting dimeric ECDs for k_(off) measurement(180 s). The interacting dimeric Nectin ECD proteins were used at 5-6concentrations of 3-fold concentration series. Data were analyzed usingOctet® Data Analysis Software 9.0 (ForteBio, Inc.) and fitted to the 1:1binding model. Equilibrium dissociation constants (K_(D)) werecalculated by the ratio of k_(on) to k_(off). Data are provided in Table5 below. hzD6.2C mAb showed specific binding to human Nectin-4 ECD,while no detectable binding was observed between hz3E2-4 mAb andNectin-1, -2, and -3 ECDs.

TABLE 5 Specificity of hzD6.2C mAb Binding to Nectin-4 Antibody ECDK_(D) (with avidity) hzD6.2C huNectin-1 ECD- — hFc huNectin-2 ECD- — hFchuNectin-3 ECD- — hFc huNectin-4 ECD- hFc <1 pM — No binding detected

Example 8 Anti-Nectin-4 Antibody Blocking of TIGIT Binding to Nectin-4Expressing Tumor Cells

Anti-Nectin-4 antibody was examined to determine whether it could blockbinding of TIGIT to Nectin-4 expressing T24 tumor cells. Stable Nectin-4expression in a T24 clone was achieved by Nectin4-lentiviraltransduction. Recombinant human TIGIT-Fc chimera was purchased from R &D Systems. Briefly, cell binding was examined on Nectin-4 expressing T24cells, which were dispensed into wells of V-bottom 96-well assay plates(1×10⁵ cells/well), spun down, and the supernatant discarded. The cellswere incubated with an anti-Nectin-4 antibody (hzD6.2C-mIgG2a) or anisotype control (mIgG2a) at eight different concentrations, startingwith 400 nM and then 1:2 serial dilution, incubated at 4° C. for 30min., and then rhTIGIT-Fc was added to each well at a finalconcentration of 40 nM. The treated cells were incubated at 4° C. for 30min., washed three times, and then commercial PE anti-human Fc as asecondary antibody (1:200 dilution) was added and incubated at 4° C. for20 min. The cells were washed twice and analyzed on BD Celesta™ flowcytometry. Percent of inhibition of TIGT (40 nM) binding due toanti-Nectin-4 antibody present was measured.

FIG. 6 shows that that anti-Nectin-4 antibody hzD6.2C-mIgG2a was capableof blocking more than 70% TIGIT binding at various concentrations, whilemIgG2a isotype did not block TIGIT binding to Nectin-4 (the measurementsslightly above or below the x-axis were within the margin of error).

Example 9 Anti-Nectin-4 Antibody Blocking of TIGIT Binding to Nectin-4Expressing Tumor Cells

To examine the in vivo efficacy of the anti-Nectin-4-TLR7 agonistconjugate surrogate (D6C mIgG2a-Compound 4.1), 7-9 week old femaleBalb/c mice (The Jackson Laboratory) were orthotopically implantedsubcutaneously into the mammary fat pad with about 1×10⁵ humanNectin-4-expressing EMT6 syngeneic breast cancer cells. Tumor volumeswere measured using the equation: VOL=(L×W²)/2. When tumors reachedapproximately 90 mm³ (Day 0), mice were sorted into groups of 8 miceeach and dosed subcutaneously with 10 mg/kg of one of: (a) mouse IgG2a(mIgG2a) isotype control, (b) D6C mIgG2a (unconjugated control), or (c)D6C mIgG2a-Compound 4.1, every 7 days for a total of 6 doses. Tumorvolume was recorded 3 times per week and mice were euthanized whentumors reached 1500 mm³.

FIGS. 7A to 7C display the anti-tumor efficacy induced by single agentanti-Nectin-4-TLR7 agonist conjugate surrogate (D6C mIgG2a-Compound4.1). This treatment inhibited tumor growth in 62.5% of mice, anddurable growth control was observed to at least day 87.

Example 10 In Vivo Pharmacodynamic Activity of Anti-Nectin4-TLR8 AgonistConjugate in Non-Human Primate (NHP)

To examine safety and tolerability, an anti-Nectin-4-TLR8 agonistconjugate was administered by subcutaneous (SC) injection to cynomolgusmonkeys at dose levels ranging from 0.1 to 12 mg/kg. Blood samples wereobtained prior to dosing and at 6, 24, 48, and 96 hours post-dose, andserum was recovered and stored in a freezer set to maintain −80° C.Samples were examined for chemokine serum concentrations using amultiplex immunooncology assay (Meso Scale Diagnostics, Rockville, Md.).In this study, the anti-Nectin-4-TLR8 agonist conjugate used was D6.2CIgG1-Compound 2.14.

Consistent with activation of TLR8, SC doses of the anti-Nectin-4-TLR8agonist conjugate led to transient increases in the serum concentrationof markers indicative of myeloid cell activation in NHP, such asmonocyte chemoattractant protein-1 (MCP-1) and interferon gamma-inducedprotein 10 (IP-10), which showed dose-response relationships,particularly at the lower dose levels (FIG. 8).

The data demonstrate that the anti-Nectin-4-TLR8 agonist conjugateactivated myeloid cells in NHP following systemic administration viasubcutaneous injection in a dose-dependent manner, and displayed afavorable safety and tolerability profile.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. A myeloid cell agonist conjugate or a saltthereof, wherein the conjugate is represented by Formula (I):

wherein: A is an anti-Nectin-4 antibody, or an antigen-binding fragmentthereof, comprising a heavy chain variable region (VH) and a light chainvariable region (VL), wherein the VH comprises a CDR1 (VH-CDR1)comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2 comprisingthe amino acid sequence of SEQ ID NO:2, a VH-CDR3 comprising the aminoacid sequence of SEQ ID NO:3; and the VL comprises a CDR1 (VL-CDR1)comprising the amino acid sequence selected from any one of SEQ IDNOS:4-6, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:7,and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:8; L is alinker; D_(x) comprises a TLR8 agonist; n is selected from 1 to 20; andz is selected from 1 to
 20. 2. The myeloid cell agonist conjugate ofclaim 1, wherein the anti-Nectin-4 antibody or antigen-binding fragmentthereof comprises: (a) a VH comprising an amino acid sequence that hasat least 90% identity with the amino acid sequence of SEQ ID NO: 10, anda VL comprising an amino acid sequence that has at least 90% identitywith the amino acid sequence of SEQ ID NO: 14; or (b) a VH comprising anamino acid sequence that has at least 90% identity with the amino acidsequence of SEQ ID NO: 10, and a VL comprising an amino acid sequencethat has at least 90% identity with the amino acid sequence of SEQ IDNO: 13; or (c) a VH comprising an amino acid sequence that has at least90% identity with the amino acid sequence of SEQ ID NO: 10, and a VLcomprising an amino acid sequence that has at least 90% identity withthe amino acid sequence of SEQ ID NO: 12; or (d) a VH comprising theamino acid sequence of SEQ ID NO: 10, and a VL comprising the amino acidsequence of SEQ ID NO: 14; or (e) a VH comprising the amino acidsequence of SEQ ID NO: 10, and a VL comprising the amino acid sequenceof SEQ ID NO: 13; or (f) a VH comprising the amino acid sequence of SEQID NO: 10, and a VL comprising the amino acid sequence of SEQ ID NO: 12.3. The myeloid cell agonist conjugate of claim 1, wherein A is ananti-Nectin-4 antibody and the antibody comprises: (a) a heavy chaincomprising an amino acid sequence that is at least 90% identity with theamino acid sequence of SEQ ID NO:24, and a light chain comprising anamino acid sequence that has at least 90% identity with the amino acidsequence of SEQ ID NO:28; or (b) a heavy chain comprising an amino acidsequence that is at least 90% identity with the amino acid sequence ofSEQ ID NO:24, and a light chain comprising an amino acid sequence thathas at least 90% identity with the amino acid sequence of SEQ ID NO:27;or (c) a heavy chain comprising an amino acid sequence that is at least90% identity with the amino acid sequence of SEQ ID NO:24, and a lightchain comprising an amino acid sequence that has at least 90% identitywith the amino acid sequence of SEQ ID NO:26; or (d) a heavy chaincomprising an amino acid sequence of SEQ ID NO:24, and a light chaincomprising the amino acid sequence of SEQ ID NO:28; or (e) a heavy chaincomprising an amino acid sequence of SEQ ID NO:24, and a light chaincomprising the amino acid sequence of SEQ ID NO:27 or (f) a heavy chaincomprising an amino acid sequence of SEQ ID NO:24, and a light chaincomprising the amino acid sequence of SEQ ID NO:26.
 4. The myeloid cellagonist conjugate of claim 1, wherein the TLR8 agonist is selected fromany one of compounds 1.1-1.69 or a salt thereof.
 5. The myeloid cellagonist conjugate of claim 4, wherein the TLR8 agonist is compound 1.36,1.50, 1.57, 1.60, or 1.64 or a salt thereof.
 6. The myeloid cell agonistconjugate of claim 1, wherein the TLR8 agonist is:


7. The myeloid cell agonist conjugate of claim 1, wherein the linker isrepresented by formula (V):

wherein L⁴ represents the C-terminus of the peptide and L⁵ is selectedfrom a bond, alkylene and heteroalkylene, wherein L⁵ is optionallysubstituted with one or more groups independently selected from R³²; RX*comprises a bond, a succinimide moiety, or a hydrolyzed succinimidemoiety bound to a residue of the antibody, wherein

 on RX* represents the point of attachment to the residue of theantibody and the other

 represents the point of attachment to the TLR8 agonist; and R³² isindependently selected at each occurrence from halogen, —OH, —CN,—O—C₁₋₁₀ alkyl, —SH, ═O, ═S, —NH₂, —NO₂; C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, andC₂₋₁₀ alkynyl, each of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl isoptionally substituted with one or more substituents independentlyselected from halogen, —OH, —CN, —O—C₁₋₁₀ alkyl, —SH, ═O, ═S, —NH₂, and—NO₂.
 8. The myeloid cell agonist conjugate of claim 7, wherein thepeptide of the linker is Val-Cit or Val-Ala.
 9. The myeloid cell agonistconjugate of claim 7, wherein the linker is a cleavable linker.
 10. Themyeloid cell agonist conjugate of claim 1, wherein L-D_(x) has astructure selected from any one of:

and a salt of any one thereof, wherein the RX* is a bond, a succinimidemoiety, or a hydrolyzed succinimide moiety bound to a residue of theantibody, and wherein

 on RX* represents the point of attachment to the residue of theantibody.
 11. The myeloid cell agonist conjugate of claim 10, wherein: nis an integer from 1 to about 10, or from 1 to about 5, or is 1 or 2, oris 1; and z ranges from 1 to about 10, or from 1 to about 9, or from 1to about 8, or 2 to about 6, or about 3 to about 5, or about
 4. 12. Themyeloid cell agonist conjugate or a salt thereof of claim 1, wherein: Ais an anti-Nectin-4 antibody comprising: (a) a heavy chain comprising anamino acid sequence that is at least 90% identity with the amino acidsequence of SEQ ID NO:24, and a light chain comprising an amino acidsequence that has at least 90% identity with the amino acid sequence ofSEQ ID NO:28; or (b) a heavy chain comprising an amino acid sequencethat is at least 90% identity with the amino acid sequence of SEQ IDNO:24, and a light chain comprising an amino acid sequence that has atleast 90% identity with the amino acid sequence of SEQ ID NO:27; L-D_(x)has a structure selected from any one of:

 wherein RX* is a bond, a succinimide moiety, or a hydrolyzedsuccinimide moiety bound to a residue of the antibody, wherein

 on RX* represents the point of attachment to a cysteine residue of theantibody; n is 1; and z ranges from 2 to about
 6. 13. The myeloid cellagonist conjugate or a salt thereof of claim 12, wherein A is ananti-Nectin-4 antibody comprising a heavy chain comprising an amino acidsequence of SEQ ID NO:24 and a light chain comprising the amino acidsequence of SEQ ID NO:28, and L-D_(x) has a structure of:


14. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:27, and L-D_(x) has astructure of:


15. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:28, and L-D_(x) has astructure of:


16. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:27, and L-D_(x) has astructure of:


17. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:28, and L-D_(x) has astructure of:


18. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:27, and L-D_(x) has astructure of:


19. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:28, and L-D_(x) has astructure of:


20. The myeloid cell agonist conjugate or a salt thereof of claim 12,wherein A is an anti-Nectin-4 antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:24 and a light chaincomprising the amino acid sequence of SEQ ID NO:27, and L-D_(x) has astructure of:


21. A pharmaceutical composition comprising the myeloid cell agonistconjugate of claim 1 and a pharmaceutically acceptable excipient. 22.The pharmaceutical composition of claim 21, wherein the average TLR8agonist-to-antibody ratio of the conjugate ranges from about 2 to about8, about 3 to about 8, about 3 to about 7, or about 3 to about
 5. 23. Apharmaceutical composition comprising the myeloid cell agonist conjugateof claim 12 and a pharmaceutically acceptable excipient.
 24. Thepharmaceutical composition of claim 23, wherein the average TLR8agonist-to-antibody ratio of the conjugate ranges from about 2 to about8, about 3 to about 8, about 3 to about 7, or about 3 to about
 5. 25. Amethod of treating a Nectin-4 expressing cancer, comprisingadministering to a subject in need thereof an effective amount of amyeloid cell agonist conjugate of the pharmaceutical composition ofclaim
 23. 26. The method of claim 25, wherein the cancer is one or moreof bladder cancer, breast cancer, lung cancer, head and neck cancer,cervical cancer, and pancreatic cancer.
 27. The method of claim 26,wherein the bladder cancer is urothelial cancer; the breast cancer istriple-negative breast cancer; the lung cancer is non-small cell lungcancer, squamous cell carcinoma, or lung adenocarcinoma; the head andneck cancer is head and neck squamous cell carcinoma; the cervicalcancer is cervical squamous cell carcinoma or endocervicaladenocarcinoma; and the pancreatic cancer is pancreatic adenocarcinoma.28. The method of claim 25, wherein the method comprises administeringan additional therapeutic agent selected from a small moleculeinhibitor, a CAR-T cell, a chemotherapeutic, a therapeutic antibody, andan antibody-drug conjugate.
 29. The method of claim 28, wherein theadditional therapeutic agent is an anti-TIGIT antibody or an adenosinereceptor antagonist.
 30. The method of claim 25, wherein the methodfurther comprises administering an anti-PD-1 or anti-PD-L1 antibody.